The newly discovered oocyte-derived growth factor BMP15, like GDF9, is a member of the transforming growth factor-£] (TGF-£]) superfamily. To our knowledge, however, the expression and function of BMP15 in ovarian tissues has not yet been studied in the pig. We asked whether the relative abundance (RA) of BMP15 mRNA changes in oocytes and follicular cells during pre-ovulatory period or culture of cumulus-oocyte-complexs (COCs) in vitro. Denuded oocytes, cumulus cells and COCs were isolated from growing and pre-ovulatory follicles. Total RNA was extracted from the cells, and reverse transcriptase polymerase chain (RT-PCR) was carried out using specific oligo-nucleotide primers. Expansion of COCs was firstly observed at 18 h, when this period we found BMP15 mRNA expression obviously. But when we culture of denuded oocytes instead of COCs, BMP15 mRNA didn¡¦t express throughout the period. We also detected GDF9 and BMP15 mRNA in pig embryonic stage and in differentiation stage of pig stem cell. GDF9 mRNA level continued to express after fertilization, but BMP15 mRNA didn¡¦t appear. We also added BMP15 antibody against Expanding of COCs. The present results support the concept that BMP15 is a key mediator of oocyte-enabled cumulus expansion in pig.

Research on in vitro embryo production (IVP) in the equine is impeded by the limited availability of mature oocytes as the mare is mono ovulating and superovulation is still difficult (Dippert and Squires, 1994; Bezard et al., 1995; Alvarenga et al., 2001b). Despite recent improvement in IVM of equine oocytes, success rates of IVM in that species remain low in all culture media tested compared to other species (Goudet et al., 2000b). However, most studies have focused on the percentage of oocytes reaching the metaphase II stage (nuclear maturation) but few concentrated on the final oocyte competence as measured by its ability to develop into a blastocyst and further establish a pregnancy. Blastocyst production rate is influenced not only by culture environment but also by oocyte maturation conditions. Under in vitro culture conditions, oxidative modifications of cell components via increased ROS represent a major culture induced stress (Johnson and Nasr-Esfahani, 1994). Anti-oxidant systems can attenuate the deleterious effects of oxidative stress by scavenging ROS (Del Corso et al., 1994). Glutathione, a tripeptide thiol, is the major non-protein sulfydryl compound in mammalian cells that plays an important role in protecting the cell from oxidative damage (Meister and Tate, 1976; Meister and Anderson, 1983). It has been suggested that GSH content in oocytes may serve as a reservoir protecting the zygote and the early embryos from oxidative damage before genomic activation and de novo GSH synthesis occur (Furnus et al., 1998; de Matos and Furnus, 2000). The addition of GSH synthesis precursors, such as cysteamine, a thiol compound, to IVM media has been shown to improve IVP in various species (Takahashi et al., 1993; de Matos et al., 1995; Grupen et al., 1995; de Matos et al., 2002a; de Matos et al., 2002b; de Matos et al., 2003; Gasparrini et al., 2003; Oyamada and Fukui, 2004; Balasubramanian and Rho, 2007; Anand et al., 2008; Singhal et al., 2008; Zhou et al., 2008). Very little information on the use of thiol compounds in the equine is available. Conventional in vitro fertilization (IVF) has not been successful in the mare, and a repeatable IVF technique has not yet been developed (Alm et al., 2001). To overcome the limitation of conventional IVF procedures, other methods to produce embryos from oocytes, either in vivo or in vitro, have been investigated. Among these, intra cytoplasmic sperm injection (ICSI) has permitted efficient equine in vitro blastocyst production (Galli et al., 2002; Lazzari et al., 2002; Choi et al., 2006a; Choi et al., 2006c). However, ICSI requires specific equipment and skills. Transfer of an immature oocyte into the preovulatory follicle of an inseminated recipient mare (Intra-Follicular Oocyte Transfer, IFOT) has produced embryos but the success rate was low (Hinrichs and Digiorgio, 1991). Similarly, oocyte transfer (OT) into the oviduct of an inseminated recipient mare was investigated (McKinnon et al., 1988; Carnevale, 1996; Hinrichs et al., 1997; Carnevale et al., 2001; Carnevale et al., 2003; Carnevale, 2004), and commercial programs using OT for mares with reproductive abnormalities are now available (Carnevale et al., 2001). Unfortunately, IFOT is poorly documented in the literature and reports of OT have been published by various laboratories and under various conditions, making comparisons between results and choosing among these as substitutive techniques to ICSI or embryo transfer difficult. The first aim of the present work was to investigate if there is an influence of supplementation with 100 µM of cysteamine on conventional IVF success rate. Cumulus oocytes complexes (COCs) retrieved by transvaginal ultrasound guided aspiration were matured in vitro with or without cysteamine supplementation and were then submitted to conventional IVF using either calcium ionophore or heparin as capacitation treatment for spermatozoa. A total of 131 oocytes were evaluated for evidence of sperm penetration. Both techniques (ionophore or heparin) yielded 6% of IVF and results were similar both for the cysteamine and the control group. This success rate of IVF is low compared to some published data (Palmer et al., 1991; Dell'Aquila et al., 1996; McPartlin et al., 2009) but similar to what others reported in the literature (Choi et al., 1994; Dell'Aquila et al., 1997a). Although, it seems likely that cysteamine did not significantly improve IVF rates under our conditions, our general success rates for IVF procedures may be too low for us to conclude definitely about the effect of cysteamine. As ICSI was not available to us, the second aim of this work was to determine what in vivo technique could best bypass the lack of an efficient conventional IVF procedure. We compared embryo production following transfer of in vivo recovered oocytes (1) into a recipients oviduct or (2) into her preovulatory follicle either immediately after ovum pick up or (3) after in vitro maturation. Recipients were inseminated with fresh semen of a stallion with a known normal fertility. Ten days after transfer, rates of embryos collected in excess to the number of ovulations were calculated and compared for each group. Embryo collection rates were 32.5% (13/40), 5.5% (3/55) and 12.8% (6/47) for OT, post-IVM and immediate IFOT respectively. OT significantly yielded more embryos than immediate and post-IVM IFOT did. These results show that, when ICSI is not an option, intra-oviductal oocyte transfer is to be preferred to IFOT, as an in vivo alternative, to bypass the inadequacy of conventional in vitro fertilization and to assess oocyte developmental competence. After it was established that in comparison to IFOT, OT is the most reliable in vivo alternative to in vitro fertilization where ICSI technology is not available, this technique was used to assess the effect of cysteamine supplementation on nuclear maturation and oocyte competence. The third aim of this work was to investigate the influence of supplementation with 100 µM of cysteamine on in vitro nuclear and cytoplasmic maturation by specific DNA staining and the ability of oocytes to undergo in vivo fertilization after OT. Oocytes were collected by transvaginal ultrasound guided aspiration and matured in vitro with (cysteamine group) or without (control group) cysteamine. The nuclear stage after DNA Hoechst staining and the embryo yield following OT were used as a criterion for assessing nuclear and cytoplasmic maturation, respectively. Overall maturation rate was 52%, which is rates reported in the literature ranging from 40 to 70% in the equine (Goudet et al., 1997a; Bogh et al., 2002; Hinrichs et al., 2005; Galli et al., 2007). Nuclear maturation was not statistically different (p>0.05) between oocytes cultured with or without cysteamine (55% and 47% respectively). From 57 oocytes transferred to the oviduct in each group, the number of embryos collected was 10 (17%) in the control group and 5 in the cysteamine group (9%). Those two percentages were not statistically different (p>0.05). Contrary to the data described in other domestic species, there was no effect of cysteamine on in vitro nuclear maturation, or in vivo embryonic development under our conditions. Under our conditions, the addition of 100 µM of cysteamine to a classic culture medium does not improve equine oocyte maturation or embryonic development after OT. The same dose failed to increase GSH content in the equine (Luciano et al., 2006). However, the effect of cysteamine supplementation is highly species and concentration dependant. The inadequacy of the chosen concentration may explain that equine embryo production has not been increased by the cysteamine under our conditions as opposed to what has been observed in many other species. Alternatively, we can hypothesize that some substances present in the IVM medium can interfere with GSH synthesis. This has been suggested for FSH and estradiol (Bing et al., 2001) and, although our maturation medium is not supplemented with gonadotropins or estradiol, factors contained in fetal calf serum or EGF might also have an effect on GSH synthesis. Considering its beneficial effects in many other species, supplementation with cysteamine to different IVM media should be further investigated in the equine. Ideally combining different concentrations and ICSI or OT in order to determine an optimal concentration and its effects on oocyte developmental competence.

Pre-pubertal pig oocytes possess lower developmental competence than those from adult pigs following in vitro maturation (IVM). Previous studies have demonstrated that exposure of pre-pubertal oocytes to 1 mM dibutyryl cAMP (dbcAMP), a membrane permeable cyclic adenosine monophosphate (cAMP) analogue, for the first 20 h of IVM improves the rate of blastocyst development. Developmental competence of in vitro matured pig oocytes has been reported to increase with increasing follicle size. In this thesis, experiments were carried out using pre-pubertal and adult pig oocytes to investigate the relationship between donor age, intra-oocyte cAMP level and follicle size in terms of oocyte maturation and developmental competence. These experiments demonstrated that, while ovarian, follicular and oocyte morphology are immediately altered with the onset of puberty, pre-pubertal oocytes must be exposed to more than the first oestrous cycle to achieve improved developmental competence in vitro. Later experiments demonstrated that pre-pubertal oocytes accumulate less cAMP during IVM, undergo more rapid meiotic progression and display reduced rates of blastocyst development compared to in vitro matured adult oocytes. Treatment with dbcAMP for 22 h IVM increased the cAMP content of pre-pubertal oocytes, slowed meiotic progression during IVM and improved the rate of blastocyst formation. While the cAMP concentration of pre-pubertal oocytes was increased to levels similar to that of adult oocytes, rates of blastocyst formation remained lower, suggesting that additional factor(s) are required for oocyte maturation. This thesis also examined the follicle size cohorts that make up the 3-8 mm aspiration range on pig ovaries. The surface of pre-pubertal ovaries contained around double the number of 3 mm follicles compared with adult ovaries. Blastocyst development of pre-pubertal oocytes increased with increasing follicle size and was highest using oocytes from 5-8 mm follicles, while adult oocytes from all follicle size cohorts displayed similar high rates of blastocyst formation. The interaction between follicle size and cAMP content in pre-pubertal oocytes was examined next. Cumulus-oocyte complexes (COCs) from 3 mm follicles accumulated less intra-oocyte and inter-COC cAMP and displayed reduced cumulus expansion compared with COCs from 5-8 mm follicles. While dbcAMP treatment increased the cAMP content of oocytes from 3 mm follicles, it had no effect on the cAMP content of the whole COC. These findings suggest that inadequate levels of intra-oocyte cAMP during IVM contribute to the low developmental competence of pre-pubertal oocytes from 3 mm follicles, suggesting that cAMP transfer, production or degradation processes are incomplete. Analysis of steroid content from different follicle size cohorts revealed that the progesterone content of prepubertal follicular fluid (FF) increased with increasing follicle size, yet overall was lower than that of adults. This suggests that differences may exist in the gonadotropinstimulated steroidogenic activity of granulosa cells of pre-pubertal COCs from different follicle sizes. Since progesterone secretion did not differ between pre-pubertal and adult COCs, it appears that the downstream pathway from the granulosa cell response rather than the actual quantity of progesterone is important for subsequent maturation processes. These studies then examined gap junction communication (GJC) within the pre-pubertal COC during IVM to examine whether the positive effects of increasing follicle size and dbcAMP on intra-oocyte cAMP levels relates to improved cAMP transfer between the cumulus cell layer and oocyte. Cumulus cell-oocyte GJC during IVM was maintained for a longer period in pre-pubertal COCs from 3 mm follicles than in those from 5-8 mm follicles. Treatment with dbcAMP had minimal effect on GJC in either COC type, thus the dbcAMP-induced increase in intra-oocyte cAMP levels appears independent of GJC. Differences in GJC during IVM together with the COCs ability to increase intraoocyte cAMP levels during IVM, suggests that differences may exist in the quantity of gonadotropin receptors, which are responsible for cAMP production, within the cumulus layer of COCs from 3 mm compared with 5-8 mm follicles. In conclusion, this thesis has demonstrated that an increase in intra-oocyte cAMP is necessary during maturation for completion and synchronisation of maturation and high developmental competence of the pig oocyte. Comparison of 3, 4 and 5-8 mm follicle sizes in the pre-pubertal pig, as described here, provides an excellent model for further investigation into the role of cAMP and the other factors required for co-ordination of oocyte nuclear and cytoplasmic maturation and subsequent embryo production. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1297309 / Thesis (Ph.D.) -- School of Paediatrics and Reproductive Health, 2007

The ability to efficiently analyze changing data is a key requirement of many real-time analytics applications. Traditional approaches to this problem were developed around the notion of Incremental View Maintenance (IVM), and are based either on the materialization of subresults (to avoid their recomputation) or on the recomputation of subresults (to avoid the space overhead of materialization). Both techniques are suboptimal: instead of materializing results and subresults, one may also maintain a data structure that supports efficient maintenance under updates and from which the full query result can quickly be enumerated. In two previous articles, we have presented algorithms for dynamically evaluating queries that are easy to implement, efficient, and can be naturally extended to evaluate queries from a wide range of application domains. In this paper, we discuss our algorithm and its complexity, explaining the main components behind its efficiency. Finally, we show experiments that compare our algorithm to a state-of-the-art (Higher-order) IVM engine, as well as to a prominent complex event recognition engine. Our approach outperforms the competitor systems by up to two orders of magnitude in processing time, and one order in memory consumption.

雲端化的時代，傳統機械式自動販賣機漸漸跟不上人們的需求，連結網路並隨時將資料回傳雲端的智慧售貨機於焉誕生！(SRT智慧零售雜誌夏季號) 物聯網(IOT)讓新一代的智慧型自動販賣機更個性化和充滿樂趣，從而幫助提高銷售額。透過將自動販賣機連線到雲端服務和進行數據分析，物聯網還可以改善經營並開啟新的商業模式(Embedded Innovator雜誌)。 未來，隨著國人的消費行為的改變，支付的方式以及習慣越來越日新月異，從原本的實體貨幣到所謂的塑膠貨幣─信用卡，支付的行為隨著科技的演變、雲端運算的技術不斷改變，未來一支智慧型手機將取代傳統錢包、信用卡、折價券、讀卡機等相關金融支付藉以完成帳務性交易。根據Gartner(國際研究暨顧問機構)研究資料顯示，全球行動支付的交易額在2013年達到2,354億美元，相較2012年的交易額成長了44%，其中亞太地區的交易額於2013年達到740億美元較2012年成長38%。預計將在2016年亞太地區將超越非洲成為交易額最高的地區，達1,650億美元。其中行動支付主要項目，以金融轉帳和商品購買最多，分別占總交易額的71%和21%。行動支付不僅打破以往交易的方式，根據IPSOS市調公司調查報告顯示，台灣37%智慧型手機用戶每月至少使用行動裝置購買實體商品一次，智慧型手機以及行動支付的普及推動智慧型販賣機的誕生。

Made available in DSpace on 2014-08-20T13:32:49Z (GMT). No. of bitstreams: 1 tese_elisa_caroline_da_silva_santos.pdf: 986483 bytes, checksum: 66da36a6826c54f5bd79701d8e49d914 (MD5) Previous issue date: 2014-02-18 / The production of swine embryos in vitro requires efficient in vitro maturation (IVM), which can be achieved by selection the most competent cumulus-oocyte complexes COC).The Brilliant Cresyl Blue (BCB) dye allows the selection of COC with complete growth by assessing their levels of the G6PDH enzyme. However, there is a possible negative effect of selection with BCB. This effect may be due to its intrinsic toxicity or to factors related to the composition of the media used during the test. This research had the objectives: to determine potential toxicity after exposure to BCB and to evaluate the effect of different medias for BCB staining on the ability to support oocyte development. On the first research, after BCB staining and after IVM, several tests were performed to evaluate the effects of their potential toxicity on mitochondrial activity and functionality: reactive oxygen species (ROS), ATP, mitochondrial membrane potential and the number of copies of mitochondrial DNA. The results showed that oocytes stained with BCB produced high levels of ROS, compared with control immediately after staining and after the IVM. The ATP and mitochondrial membrane potential showed similar results between groups after staining, however, after IVM oocytes BCB showed lower membrane potential and ATP. There was no difference in the number of copies of mtDNA in the evaluated groups. Already, on test of ATP content in early embryos, ATP was lower in BCB oocytes, however, there was no difference statistical. In second study, the most commonly used media, D-PBS, was compared with a more elaborate media for BCB called here: ReproPEL. The COC s submitted to both media were submitted to nuclear and cytoplasmatic maturation, parthenogenetic activation, and to the comet test. The great rates of nuclear IVM (P<0.05) were obtained for DPBS+ (63.1%), ReproPELc (55.1%) and ReproPEL+ (50.2%). The group with smaller area of CG (P<0.05), showing better migration, were ReproPELc, D-PBS+, D-PBS- and ReproPEL+. The parthenogenetic activation indicated that ReproPEL media presented satisfactory capacity of oocyte maintenance, resulting in acceptable rates of development to blastocyst stage: 13.0% for ReproPEL+; and 12.7% for ReproPELc. So, the ReproPEL media can be used for maintenance of swine oocytes, but it was not the most appropriate media for BCB staining. Moreover, after exposure to BCB and after IVM, BCB oocytes presented high toxicity at mitochondrial level, due to increased production of ROS, decreased membrane potential and compromised ATP production. However, the mitochondrial function was restored in early embryonic development. In conclusion, BCB was responsible for toxicity in immature swine oocytes, nevertheless, further studies must be performed to evaluate the changes caused by BCB in the embryonic level. / Para a obtenção de embriões suínos produzidos in vitro faz-se necessário que a maturação in vitro (MIV) ocorra de forma eficiente, o que exige a seleção dos complexos cumulus-oócitos (CCOs) mais competentes. O corante Brilliant Cresyl Blue (BCB) permite selecionar os CCOs que completaram seu crescimento, mediante a avaliação dos níveis da enzima G6PDH. Entretanto, existe um possível efeito nocivo relacionado ao processo de seleção com BCB, o qual pode ser devido a uma toxicidade intrínseca do corante ou aos vários fatores relacionados à composição dos meios para a realização do teste. Desta forma, esta pesquisa teve como objetivos: averiguar a existência de toxicidade após exposição ao BCB e avaliar o efeito de diferentes meios para a coloração com BCB sobre a capacidade de suporte ao desenvolvimento oocitário. Na primeira pesquisa, após a coloração com BCB e após a MIV, vários testes foram realizados para avaliar os efeitos de sua potencial toxicidade sobre a atividade e a funcionalidade mitocondrial: análises de espécies reativas de oxigênio (ROS), ATP, potencial de membrana mitocondrial e número de cópias de DNA mitocondrial. Como resultados, obteve-se que oócitos corados com BCB produziram altos níveis de ROS quando comparados com o controle imediatamente após a coloração e após a MIV. O ATP e potencial de membrana mitocondrial apresentaram resultado similar entre os grupos após a coloração, porém, após a MIV oócitos BCB apresentaram menor potencial de membrana e ATP. Não ocorreu diferença no número de cópias do DNAmt nos grupos avaliados. Já, no teste do conteúdo de ATP em embriões iniciais, o ATP foi inferior em oócitos BCB, porém, não ocorreu diferença significativa. Na segunda pesquisa, comparou-se o meio mais utilizado, D-PBS, com um meio mais elaborado para o BCB, chamado de ReproPEL. Os CCOs submetidos aos dois meios foram submetidos à MIV e avaliados quanto à maturação nuclear e citoplasmática, ativação partenogenética e ao teste cometa. Na MIV nuclear, as maiores taxas de MII (P<0,05) foram obtidas no DPBS+ (63,1%), ReproPELc (55,1%) e ReproPEL+ (50,2%). Quanto à densidade dos GC, os grupos com menor área (P<0,05), evidenciando melhor migração, foram ReproPELc, D-PBS+, D-PBS- e ReproPEL+. A ativação partenogenética demonstrou que o meio ReproPEL possui boa capacidade de manutenção oocitária, possibilitando taxas aceitáveis de desenvolvimento até o estágio de blastocisto: ReproPEL+ (13,0%); e ReproPELc (12,7%). Desta forma, o meio ReproPEL pode ser indicado para a manutenção oocitária, porém não foi o meio mais indicado para o corante BCB. Com relação à toxicidade, após a exposição ao BCB e após a MIV, os oócitos BCB apresentaram alterações em nível mitocondrial, devido ao aumento na produção de ROS, diminuição do potencial de membrana e ao comprometimento da produção de ATP. Porém, a função mitocondrial foi restaurada no início do desenvolvimento embrionário. Com tudo isso, conclui-se que o BCB foi responsável por toxicidade em oócitos suínos imaturos, sendo necessários novos estudos para avaliar as alterações causadas pelo BCB em nível embrionário.

La ICSI porcina es una herramienta con gran potencial aplicativo en diversos campos, entre los que destacan la producción de animales transgénicos, y la recuperación de razas en peligro de extinción. Aunque en la actualidad existen referencias de obtención de descendencia viva, el rendimiento es inferior al de otras especies, posiblemente debido al desconocimiento de las condiciones idóneas, y la dificultad de los cigotos para alcanzar el estadío de blastocisto in vitro. El presente trabajo se llevó a cabo para determinar diferentes factores que podrían afectar al rendimiento de la técnica, estudiando el efecto de 1) la secuencia de cultivo de los zigotos recién inyectados; 2) modificaciones en el sistema de MIV tradicional, y por último 3) la activación exógena del ovocito mediante la inyección de inositol trifosfato con el espermatozoide. El objetivo global de este estudio fue el de incrementar el rendimiento final de la ICSI en la especie porcina. / ICSI in pigs is a tool with an important applicable potential in diverse fields. One of this is the production of transgenic animals, and the conservation of endangered species. Even though there are some cases of living offspring, its output is still quite low comparing to other species, possibly due to unknown factors referring to ideal conditions for the development, and to the difficulty of the zygotes to reach the blastocyst stage in vitro. The goal of this study was to evaluate different factors affecting the ICSI performance. This was done by studying 1) the sequence of culture of the injected oocytes; 2) In vitro maturation (IVM) modifications, through meiotic inhibitors, such as roscovitine, and changes in IVM duration time, and finally 3) the exogenous oocyte activation through inositol triphosphate (InsP3) injection together with the sperm. The main objective of this study was to increase the final performance of ICSI in pigs.

Made available in DSpace on 2014-08-20T13:32:49Z (GMT). No. of bitstreams: 1 tese_bruna_curcio.pdf: 7472121 bytes, checksum: b7f2b517d0687e52e051d45e308fb2eb (MD5) Previous issue date: 2006-12-05 / The aim of the study was to investigate: 1) the ultrastructural morphologic characteristics in equine oocytes subjected to different times of exposure to cryoprotectant solutions and 2) the effect of initial cumulus morphology and cryoprotectants in the nuclear in vitro maturation (IVM) of the vitrified immature equine oocytes. The oocytes were obtained from ovaries from a slaughterhouse. In the first study 30 oocytes where divided in three groups: Control group (G1, n=10); Group 2 (G2, n=10), the oocytes were vitrified for exposure to VS-1 for 3min and VS-2 for 1min; Group 3 (G3, n=10), exposure to VS1 for 1.5min and VS-2 for 30sec. The oocytes were vitrified in open-pulledstraws (OPS). The ultrastructural characteristics where observed using a transmission electron microscope. The oocytes were classified as: I) oocytes morphologically normal; II) oocytes which presented intermediate damage but had completed organelles, and III) oocytes with severe morphological abnormalities. In the second study, compact (Ccp; n=248) and expanded (Cex; n=264) cumulus oocyte complexes were divided in three groups: Control, Treatment-1 (T1 - Ethylene glycol-EG + Dimetyl sulfoxide-DMSO + SIB) and Treatment-2 (T2 - Formamide + EG + DMSO + Polyvinylpyrrolidone + SIB). The control group was immediately matured in vitro, the other immature oocytes were vitrified in OPS and then matured in vitro. The maturation stage was observed under a fluorescence microscope (Hoechst 33342). The results of ultrastructural morphology were rated as Class I: 80% oocytes of G1, 30% of G2 and 60% of G3; Class II: 0% oocytes of G1, 20% of G2 and 30% of G3 30%, and Class III: 20% oocytes of G1, 50% of G2 and 10% of G3. The maturation rates (metaphase II - MII) were: 41% Control group, 38,3% T1 and 33,3% T2 (P>0,05). In the control group, the MII rates were higher in Cex oocytes (53,2%) than those Cco oocytes (29,3%; P<0,05). The initial cumulus morphology didn t significantly affect MII rates after vitrification (P>0,05). However, Cex oocytes had higher MII rates than Ccp oocytes in T1 (50% vs 27,3%) and T2 (45,7% vs 21,6%). It can be concluded that the reduction in time of exposure to cryoprotectant solutions resulted in better preservation of ultrastructural characteristics of equine oocytes submitted to vitrification. Immature equine oocytes can be IVM after vitrification/re-warming, and satisfactory MII rates can be obtained. The initial cumulus morphology did not affect nuclear in vitro maturation of equine oocytes after vitrification. / Os objetivos do presente estudo foram: 1) analisar características morfológicas ultra-estruturais em oócitos eqüinos submetidos à vitrificação; 2) avaliar a maturação nuclear in vitro (MIV) de oócitos eqüinos vitrificados em soluções contendo bloqueadores sintéticos da formação de gelo (SIB); 3) considerar a influência das características das células do cumulus-oophorus no momento da coleta sobre a maturação nuclear in vitro. Foram utilizados complexos cumulusoócito obtidos de ovários provenientes de éguas de abatedouro. No primeiro experimento 30 oócitos foram divididos em 3 grupos: Grupo controle (G1, n=10); Grupo 2 (G2, n=10), vitrificados após exposição de 3min em VS-1 e 1min em VS-2; Grupo 3 (G3, n=10), exposição por 1,5min à VS-1 e 30s à VS-2. A vitrificação foi realizada em palhetas abertas estiradas (OPS). As características ultra-estruturais foram observadas em microscópio eletrônico de transmissão, sendo os oócitos classificados em três categorias: I) Oócitos sem alterações; II) oócitos com alterações intermediárias e III) oócitos com alterações severas. No segundo experimento, oócitos cumulus compacto (Ccp; n=248) e cumulus expandido (Cex; n=264) foram divididos em três grupos: Controle, Tratamento-1 (T1 - Etilenoglicol-EG + Dimetilsulfóxido-DMSO + SIB) e Tratamento-2 (T2 - formamida + EG + DMSO + Polivinilpirrolidona + SIB). O grupo controle foi MIV imediatamente após a coleta, o restante foi vitrificado imaturo em OPS e submetido a MIV após o reaquecimento. O estágio de maturação após incubação foi avaliado em microscópio de fluorescência (Hoechst 33342). Na avaliação de ultra-estrutura foram classificados no escore I 80% oócitos G1, 30% do G2 e 60% do G3; Classificação II: 0% dos oócitos G1, 20% do G2 e 30% do G3; e Classificação III: 20% dos G1; 50% do G2 e 10% do G3. Na avaliação da maturação, o índice de oócitos que atingiram MII foi 41% Controle, 38,3% T1 e 33,3% T2 (P>0,05). Nos oócitos do grupo controle, o índice de MII foi superior em oócitos Cex (53,2%) do que oócitos Ccp (29,3%; P<0,05). A avaliação da influência das características das células do cumulus na vitrificação não detectou diferença (P>0,05). Contudo oócitos Cex obtiveram maiores índices de MII do que oócitos Ccp em T1 (50% vs 27,3%) e T2 (45,7% vs 21,6%). Pode-se concluir que a diminuição do período de exposição às soluções de vitrificação resultou em melhor preservação das características ultra-estruturais de oócitos eqüinos submetidos à vitrificação. Oócitos eqüinos imaturos podem ser MIV após vitrificação/reaquecimento, obtendo índices satisfatórios de MII. As características das células do cumulus, no momento da coleta, não interferem no índice de maturação nuclear in vitro de oócitos eqüinos após vitrificação.

Made available in DSpace on 2014-08-20T13:32:50Z (GMT). No. of bitstreams: 1 dissertacao_caroline_gomes_lucas.pdf: 894020 bytes, checksum: 8d27baa8c8ae08744efe112062ff8b86 (MD5) Previous issue date: 2014-02-19 / The possibility of increasing the genetic pattern and animal production make the in vitro production of embryos an extremely valuable technique for the technological development of livestock. However, due to the need for mimicking the in vivo process, that consists in several interdependent steps there are difficulties in setting the optimal conditions for each situation performed. The improvement of in vitro maturation (IVM) protocols through supplementation with different molecules can increase the efficiency of the culture medium and improve the competence of oocytes for fertilization and embryogenesis. A promising approach is the realization of mediated delivery of nanocarriers molecules. Tretinoin (TTN, all- trans retinoic acid - ATRA ) is a natural retinoid and active metabolite of vitamin A with important action in cell proliferation and differentiation and embryonic development under both in vivo and in vitro conditions. TTN acts on the cytoplasmic maturation process, in the initial embryonic development and oocyte competence, improving the quality of embryos generated. The combinations of tretinoin with polymeric nanoparticles are alternatives to enhance the solubility and chemical stability of this molecule, allowing controlled release and decreased degradation. The objective of this study was to evaluate the effects of IVM medium supplementation with tretinoin-loaded lipid-core nanocapsules (TTN-LNC) at concentrations of 0.25, 0.5 and 1 μM, by analysis of embryonic development until the blastocyst stage, production of reactive oxygen species (ROS), and expression of genes related to apoptosis and pluripotency. As main results, TTN-LNC 0.25 μM increased the rate of blastocyst production and reduced ROS production. In addition, TTN and TTN-LNC induced a lower gene expression of Bax and SHC1, suggesting beneficial effects on the development of embryos. The results indicate that nanoencapsulation allows the use of a lower dose of TTN-LNC with consequent obtention of higher percentages of blastocyst production and decreased production of ROS, making nanoembriology a potential tool for improving bovine embryos IVP. / A possibilidade de aumento do padrão genético e da produção animal torna a produção in vitro (PIV) de embriões uma técnica extremamente valiosa para o desenvolvimento tecnológico da pecuária. No entanto, devido a necessidade de uma mimetização do processo in vivo, que consiste em várias etapas interdependentes, há dificuldades em ajustar as condições ótimas requeridas para cada situação reproduzida. O melhoramento dos protocolos de maturação in vitro (MIV) através da suplementação com diferentes moléculas permite aumentar a eficiência dos meios de cultivo e melhorar a competência de oócitos para a fertilização e embriogênese. Uma abordagem altamente promissora é a realização da entrega de moléculas mediada por nanocarreadores. A tretinoína (TTN, ácido retinóico all-trans - ATRA) é um retinóide natural e metabólito ativo da vitamina A, com ação importante na proliferação e diferenciação celular, e no desenvolvimento embrionário tanto em condições in vivo como in vitro. A TTN age no processo de maturação citoplasmática, no desenvolvimento inicial embrionário e competência oocitária melhorando a qualidade dos embriões gerados. A associação da tretinoína à nanopartículas poliméricas surge como alternativa para melhorar a solubilidade e estabilidade química desta molécula, possibilitando uma liberação controlada e redução da degradação. O objetivo deste trabalho foi avaliar os efeitos da suplementação com nanocápsulas de núcleo lipídico associadas à tretinoína (TTN-LNC) no meio de MIV, nas concentrações de 0,25, 0,5 e 1 μM, através da análise do desenvolvimento embrionário até o estágio de blastocisto, produção de espécies reativas de oxigênio (EROs) e expressão de genes relacionados a apoptose e pluripotência. Como principais resultados, a TTN-LNC a 0,25 μM aumentou a taxa de produção de blastocistos e reduziu a produção de EROs. Além disso, TTN e TTN-LNC induziram uma menor expressão dos genes Bax e SHC1 sugerindo efeitos benéficos sobre o desenvolvimento embrionário. Os resultados indicam que a nanoencapsulação permite a utilização de uma menor dose de TTN-LNC com consequente obtenção de maiores porcentagens de produção de blastocistos e diminuição da produção de EROs, tornando a nanoembriologia uma ferramenta potencial para a melhora da técnica de PIV de embriões bovinos.

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