A significant proportion of potential lambs are lost (commonly 15-20%) between ovulation and day 30 of gestation. Moreover, little is known about factors associated with multiple birth capacity of the uterus which would be necessary to convert gains in ovulation rate to the birth of live lambs. This project has investigated the relationship between maternal uterine and hormonal environment as well as the heritability of embryonic survival (ES) in prolific ewes. Litter size (LS) from known ovulation rate (OR) records (n=6393) collected over 16 years were analysed for heritability. ASReml analysis reported ES to be a trait of low repeatability (r� = 0.103) and heritability (h� = 0.04) which is consistent with earlier studies of this trait. However, pedigrees of outlier animals indicated a segregation pattern consistent with a single autosomal gene with a major affect on enhanced ES. From this flock, closely related high ovulation rate ewes with significantly different litter sizes (High ES; OR2.6/LS2.4 versus Low ES; OR2.9/LS1.6) were selected for further study. The anatomy and gene expression of the uterus collected at day 14 of the oestrous cycle (n=5 High and n=5 Low ES ewes) and day 16 of gestation (n=14 high and n=10 Low ES ewes) as well as systemic concentrations of hormones indicative of uterine (activin-A, follistatin) and ovarian (inhibin-α, progesterone) function during the oestrous cycle and early gestation were compared. Progesterone concentrations were found to rise earlier in high ES ewes with a difference in number of ewes with detectable levels of progesterone apparent by day 4 of gestation. The peak concentration and slope of progesterone increase as well as plasma profiles of oestradiol and inhibin-α were not different between groups. A number of pathways worthy of closer investigation were implicated by microarray analysis with Ingenuity Pathway Analysis, Pubmatrix, and candidate gene approaches. In particular, the altered expression of many immune cell factors suggests that high ES ewes have maternal gene expression of the inflammatory pathways favourable to embryo implantation. The plasma concentration of activin, but not follistatin, was found to be significantly higher in low ES ewes, a difference that remained apparent when the concentration of follistatin was corrected for individual samples. Furthermore, the concentration of activin, but not follistatin, was significantly elevated on day 16 of gestation in the uterine fluid of low ES ewes. Further investigation of the pattern of gene expression during the oestrous cycle and early gestation (day10-16 oestrus and days10-20 gestation) revealed that a significant increase in follistatin mRNA in the luminal epithelia and interacting trophoblast cells of the embryo occurs on day 18 and 20 of gestation. It is likely the appropriate balance between activin and follistatin during the time of implantation enhances embryonic survival in this line of ewes. This may be secondary to or concomitant with the observed earlier rise in progesterone concentration. The implication that embryo survival may be positively influenced by a single autosomal gene has important implications for New Zealand's agricultural industry.

A significant proportion of potential lambs are lost (commonly 15-20%) between ovulation and day 30 of gestation. Moreover, little is known about factors associated with multiple birth capacity of the uterus which would be necessary to convert gains in ovulation rate to the birth of live lambs. This project has investigated the relationship between maternal uterine and hormonal environment as well as the heritability of embryonic survival (ES) in prolific ewes. Litter size (LS) from known ovulation rate (OR) records (n=6393) collected over 16 years were analysed for heritability. ASReml analysis reported ES to be a trait of low repeatability (r� = 0.103) and heritability (h� = 0.04) which is consistent with earlier studies of this trait. However, pedigrees of outlier animals indicated a segregation pattern consistent with a single autosomal gene with a major affect on enhanced ES. From this flock, closely related high ovulation rate ewes with significantly different litter sizes (High ES; OR2.6/LS2.4 versus Low ES; OR2.9/LS1.6) were selected for further study. The anatomy and gene expression of the uterus collected at day 14 of the oestrous cycle (n=5 High and n=5 Low ES ewes) and day 16 of gestation (n=14 high and n=10 Low ES ewes) as well as systemic concentrations of hormones indicative of uterine (activin-A, follistatin) and ovarian (inhibin-α, progesterone) function during the oestrous cycle and early gestation were compared. Progesterone concentrations were found to rise earlier in high ES ewes with a difference in number of ewes with detectable levels of progesterone apparent by day 4 of gestation. The peak concentration and slope of progesterone increase as well as plasma profiles of oestradiol and inhibin-α were not different between groups. A number of pathways worthy of closer investigation were implicated by microarray analysis with Ingenuity Pathway Analysis, Pubmatrix, and candidate gene approaches. In particular, the altered expression of many immune cell factors suggests that high ES ewes have maternal gene expression of the inflammatory pathways favourable to embryo implantation. The plasma concentration of activin, but not follistatin, was found to be significantly higher in low ES ewes, a difference that remained apparent when the concentration of follistatin was corrected for individual samples. Furthermore, the concentration of activin, but not follistatin, was significantly elevated on day 16 of gestation in the uterine fluid of low ES ewes. Further investigation of the pattern of gene expression during the oestrous cycle and early gestation (day10-16 oestrus and days10-20 gestation) revealed that a significant increase in follistatin mRNA in the luminal epithelia and interacting trophoblast cells of the embryo occurs on day 18 and 20 of gestation. It is likely the appropriate balance between activin and follistatin during the time of implantation enhances embryonic survival in this line of ewes. This may be secondary to or concomitant with the observed earlier rise in progesterone concentration. The implication that embryo survival may be positively influenced by a single autosomal gene has important implications for New Zealand's agricultural industry.

Bibliography: leaves 150-178. / xxii, 178 leaves, [17] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates the potential of the zebrafish Danio rerio to provide a convenient, fecund and cost efficient piscine model for biotechnology. The technical and biological limitations of the model are identified and ways of overcoming or circumventing these limitations explored in three areas of biotechnology: transgenesis; partial genome manipulation; and, entire genome manipulatiion. / Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics & Gynaecology, 1996?

Bibliography: leaves 150-178. / xxii, 178 leaves, [17] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates the potential of the zebrafish Danio rerio to provide a convenient, fecund and cost efficient piscine model for biotechnology. The technical and biological limitations of the model are identified and ways of overcoming or circumventing these limitations explored in three areas of biotechnology: transgenesis; partial genome manipulation; and, entire genome manipulatiion. / Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics & Gynaecology, 1996?

Thesis (MSc.(Anatomy)--Faculty of Health Sciences)-University of Pretoria, 2006. / Includes bibliographical references.

Made available in DSpace on 2015-04-09T12:28:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-11-04Bitstream added on 2015-04-09T12:48:24Z : No. of bitstreams: 1 000815814.pdf: 419662 bytes, checksum: b03fc09d9d326c880d6e914d837b278f (MD5) / Animais da raça Holandesa, criados nas regiões de clima temperado a quente apresentam baixa eficiência quanto ao número, qualidade e competência oocitária para a produção in vitro de embriões, em comparação às raças Zebuínas. Por outro lado o controle da onda folicular e a estimulação com FSH exógeno pode incrementar o número e melhorar a qualidade e competência oocitária para a produção embrionária. Com o objetivo comparativo sete novilhas, sete vacas não lactantes e sete vacas em lactação foram utilizadas em um delineamento em “crossover” foram submetidas a quatro tratamentos: T1 – Aspiração folicular ao acaso em dia aleatório do ciclo estral; T2 – Aspiração folicular dois dias após a remoção do folículo dominante; T3 - Aspiração folicular 24 horas após remoção do folículo dominante e estimulação com dose única de FSH (100 UI para novilhas e 200 UI para vacas não lactantes ou em lactação) vinte quatro horas antes da aspiração folicular; T4 – Aspiração folicular após estimulação e indução do pico de LH (inserção de implante de progestágeno, aspiração do folículo dominante e estimulação com FSH na dose de 250 UI para novilhas, 500 UI para vacas não lactantes e lactantes, subdividas em 8 doses decrescentes, indução de luteólise e retirada do implante 36 horas antes do GnRH e aspiração folicular 24 horas após a administração de GnRH). Não foi observada diferença estatística entre os tratamentos (P>0,05) quando foi avaliada a recuperação de oócitos, no entanto a categoria de novilhas obteve melhores resultados e apresentou diferença estatística (P<0,05) quando comparada com vacas lactantes e não lactantes, porém não foi observada diferença estatística entre as categorias vacas não lactantes ou vacas lactantes. Quando os oócitos viáveis foram avaliados, a categoria de novilhas apresentou melhor resultado e diferença significativa ... / Holstein cows, bred in temperate to hot climate have low efficiency on the number, quality and oocyte competence for in vitro production of embryos, compared to Zebu breeds. On the other hand the control of follicular wave and stimulation with exogenous FSH can increase the number and improve the quality and oocyte competence for embryo production. With the comparative goal seven heifers, seven non-lactating cows and seven lactating cows were used in a complete crossover were subjected to four treatments: T1 - Follicular Aspiration randomly at any time of the estrous cycle; T2 - Follicular Aspiration two days after removal of the dominant follicle; T3 - Follicular Aspiration 24 hours after removal of the dominant follicle and stimulation with single dose of FSH (100 IU to 200 IU for heifers and non-lactating cows or lactating) twenty four hours before follicular aspiration; T4 - Follicular Aspiration after stimulation and induction of the LH surge (progestagen implant insertion, aspiration of the dominant follicle and FSH stimulation at a dose of 250 IU to 500 IU for heifers and non-lactating and lactating cows, subdivided in 8 decreasing doses, induce luteolysis and removal of the implant 36 hours before of the GnRH and follicular aspiration 24 hours after GnRH administration). No statistical difference between treatments (P>0.05) was evaluated oocyte retrieval was observed, however category heifers achieved better results and statistical differences (P<0.05) compared with lactating cows and non-lactating, although no statistical difference between the categories of non-lactating cows and lactating cows was observed. When the viable oocytes were evaluated, the grade of heifers showed better results and significant difference (P <0.05) compared with lactating and non-lactating cows, and no significant difference was observed between non-lactating cows and lactating cows (P>0.05). In relation to treatments, treatment 1 (T1) differed ...

Orientador: Cezinande de Meira / Banca: Marco Antonio Alvarenga / Banca: Julio Cesar Ferraz Jacob / Resumo: O presente estudo tem como objetivo verificar a resposta ovulatória e recuperação embrionária em éguas após o tratamento com FSHp (Folltropin-V®) em diferentes momentos, com diferentes doses e na raça BH. O crescimento folicular foi acompanhado diariamente e os tratamentos (i.m. a cada 12h) foram mantidos até que o maior(es) folículo(s) atingiu 32mm, a indução da ovulação (2500 UI de hCG i.v.) foi realizada quando o maior(es) folículo(s) atingiu 35mm e a colheita de embriões no D8. No experimento 1, a dose de 25mg de FSHp foi testada em 28 éguas para determinação do melhor momento após aspiração folicular: 13mm (n=7, salina, controle), 13mm (n=7, FSHp-F13) e 20mm (n=7, FSHp-F20); ou em um dia determinado do ciclo: D6 (n=7, FSHp- D6). No experimento 2, a dose de 50mg de FSHp foi testada em 17 éguas quando o maior folículo atingiu 13mm (n=7, FSHp-13) e comparada ao grupo controle (n=10). No experimento 3, em 26 éguas BH inciou-se os seguintes tratamentos no D6: salina (n=7, controle), 25mg de FSHp (n=7, 25FSHp-D6), 12,5mg de EPE (n=7, EPE-D6) e 50 mg de FSHp (n=5, 50FSHp-D6). Usou-se a análise de variância de perfil, seguida do método de Tukey e para os dados de embriões/ovulação foi realizado o teste exato de Fisher (5% de significância). Os tratamentos com 25mg ou 50mg não promoveram aumento significativo no número de ovulações e de embriões recuperados, mas promoveu aumento no diâmetro máximo atingido por F3. O início do tratamento quando o maior folículo da onda induzida atingiu 20mm reduziu o tempo de tratamento. Conclui-se que os tratamentos com FSHp não promoveram resposta satisfatória e que o início do tratamento próximo ao desvio permitiu redução no tempo de tratamento. / Abstract: The present study aims to verify the ovulatory response and embrionary recover in mares treated at different moments with pFSH (Folltropin-V®), with different doses and in BH breeding mares. The follicular growth was daily accompanied and treatments (i.m. BID) were kept until major follicles reached 32mm, ovulation induction (2500 UI of hCH i.v.) was done when major follicles reached 35mm and embryo collection at D8. In experiment 1, 25mg of pFSH was tested in 28 mares for determination of the best moment for start of treatment after follicle ablation: 13mm (n=7, saline, control), 13mm (n=7, FSHp-F13) e 20mm (n=7, FSHp-F20); or in a previously determined cycle day: D6 (n=7, FSHp-D6). In experiment 2, 50mg of pFSH was tested in 17 mares when the largest follicle achieved 13mm (n=7, FSHp-13) and compared to a control grupo (n=10). In experiment 3, in 26 BH mares was initiated the following treatments beginning at D6: saline (n=7, control), 25mg of pFSH (n=7, 25FSHp-D6), 12,5mg of EPE (n=7, EPE-D6) e 50 mg of pFSH (n=5, 50FSHp-D6). For statistical analysis, Profile analysis followed by Tuckey method and, for embryo/ovulation, Fisher test were used (significance at 5%). Treatments with 25mg or 50mg did not promote a significative increase on ovulations and embryo recover, but increased maximum diameter of F3. The start of treatment when the largest follicle of an induced follicular wave achieved 20mm reduced time of treatment. It is concluded that pFSH treatment did not promote a satisfactory response and that beginning of treatment close to deviation allowed the reduction of treatment time. / Mestre

The monoclonal antibody Sp12 recognizes an epitope shared by a diverse group of developmentally regulated cell surface and extracellular matrix antigens which are expressed during Strongylocentrotus purpuratus development. Immunofluorescent localization reveals antigen in the cortical granules of eggs and in the hyaline layer from fertilization through to gastrula. Primary (skeletogenic) mesenchyme and two secondary mesenchyme derivatives (blastocoelar cells and pigment cells) express antigen after their release into the blastocoel and maintain it throughout the remainder of larval development. The antibody allows detailed descriptions of blastocoelar cells, a prominent yet poorly described fibroblast-like mesenchyme lineage. In adults antigen is localized to the organic matrix which invests the calcified stereom of the test and spines. Immunogold electron microscopy shows antigen in the cortical granules of eggs, and on cell surfaces, within membrane bound vesicles, and within the Golgi apparatus of mesenchyme cells. On western blots a confluent smear of antigen (Mr primarily >$180K) is present in eggs, but is resolved into seven antigen bands (Mr from 35K to >$200K) after fertilization. A prominent antigen at 140K is newly expressed coincident with mesenchyme immunoreactivity. Antigens at 140K and 120K fade as prisms develop into plutei, while an antigen at 105K appears in older larvae. In adult test six antigens are shared with larvae, and there are two novel antigens at 75K and 110K. Immunoreactivity is eliminated by digestion of samples with endoglycosidase F, is reduced by periodate oxidation, but is unaffected by boiling. Calcium-magnesium-free-seawater or 1M glycine extract a subset of antigens from dissociating embryos, leaving a complementary subset of antigens associated with cell membranes. Membrane antigens are not extracted, at 4 C, with high or low ionic strength, organic solvents, or non-ionic detergents but are solubilized by ionic detergents. Whole antibody has no effect on development of embryos cultured in it from fertilization on, nor is there a specific effect from injection of antibody into the blastocoel of developing embryos. The shape of dissociated cells cultured in Sp12 whole antibody is markedly constrained compared to that of controls, however, this difference is not seen in cells incubated in Sp12 Fab fragments. Sp12 appears to recognize a carbohydrate moiety shared by ten glycoproteins of widely variable Mr and cell membrane affinity which are differentially expressed on mesenchyme throughout S. purpuratus development. The antibody does not disrupt development in vivo, but does affect cell shape in vitro, probably by crosslinking cell surface antigens. / Graduate

Elongation of the mouse anteroposterior axis depends on stem cell-like axial progenitors including a neuromesodermal (NM) bi-fated population existing in the primitive streak and later in the tail bud. Fate mapping experiments have demonstrated these NM progenitors reside in precise locations of the embryo. At E8.5, these cells are found in the node-streak border (NSB) and anterior epiblast on either side of the primitive streak. At tail bud stages (E10.5-E13.5), these progenitors reside in the chordoneural hinge (CNH). The coexpression of the transcription factors T (brachyury) and Sox2 has been proposed as a good marker to identify NM progenitors in vertebrates. However, this cell signature has never been thoroughly assessed during mouse axis elongation. In this thesis, I performed T and Sox2 double immunofluorescent stainings on different stages of mouse embryos and reconstructed their expression domains in the 3D images to investigate the spatiotemporal dynamics of NM progenitors during axis elongation. The results show the transient existence of T+Sox2+ cells in the posterior progenitor zone, from the headfold stage (E8.0) to the end of axis elongation (E13.5, 65somites). Moreover, the number of T+Sox2+ cells increases between E8.5 and E9.5 but gradually declines afterwards. I then investigated the time points for initiation and loss of NM progenitors by performing a series of heterotopic grafting experiments. It has been previously shown that distal epiblast (Sox2+T- cells) at LS-EB stages (E7.5) are fated to become NSB cells in E8.5 embryos. However, when cells from the distal region of LS-EB stage embryos (E7.5) were grafted to E8.5 NSB, these cells contribute extensity to the notochord but not either neural tissues or paraxial mesoderm. This indicates that NM progenitors may be not yet specified before the onset of T and Sox2 coexpression, while the notochord progenitors are already specified at E7.5. The grafting experiments also show the loss of NM progenitors at E14.5 after the end of axis elongation, which coincides with the disappearance of T+Sox2+ cells in the tail. Collectively, these results indicate that T+Sox2+ cells may represent a distinct cell state that defines NM progenitors. Wnt/β-catenin signalling has been shown to play an important role in maintaining the posterior progenitor zone. However, due to the wide expression of β-catenin and the early lethality of β-catenin null embryos, the exact effect of losing β-catenin in NM progenitors is still unknown. In this study, I took advantage of the Cre-ERT2 system and grafting technique to conditionally delete β-catenin specifically in NM progenitors during ex vivo culture. The results show that Wnt/β-catenin signalling is required cell autonomously for initiating mesoderm fate choice in NM progenitors. In its absence, mesoderm fated NM progenitors convert their fate and differentiate to neural derivatives. Moreover, the interchangeability between neural and mesodermal fate only exists in NM progenitors, as the loss of β-catenin in mesoderm committed progenitors does not affect their fate choice. Using image analysis and quantification software, I also show that Wnt/β-catenin signalling is crucial for the expansion of T+Sox2+ NM progenitors during axis elongation. Due to difficult access and a limited number of NM progenitors in vivo, in vitro generated NM progenitors from pluripotent cells, such as epiblast stem cells (EpiSCs), can offer an insight into the maintenance and differentiation of NM progenitors. Since the in vivo potential of EpiSCs had never been successfully demonstrated before, I first grafted EpiSCs into postimplantation embryos and cultured them ex vivo for 24-48 hours to assess their cell integration. The results show that EpiSCs can integrate successfully in streak stage embryos (E6.5-E7.5), but not at early somite stages (E8.5), when the epiblast has lost its pluripotency. I then further investigated the in vivo potential of EpiSC derivatives. The results show that increasing Wnt signalling in EpiSCs inhibits their ability to generate anterior neural tissues in vivo, which is consistent with the previous in vitro data. Recently, it has been demonstrated that NM progenitors can be derived from EpiSCs. These in vitro derived NM progenitors can incorporate into E8.5 embryos and give rise to both neural and mesodermal derivatives. In this thesis, I show that these in vitro derived NM progenitors do not incorporate successfully in E7.5 embryos. Collectively, by combining grafting experiments with a chimeric embryo formation assay, I can identify the in vivo stage of the in vitro counterparts of the embryonic cell types.

Made available in DSpace on 2014-06-11T19:33:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2001-11-28Bitstream added on 2014-06-13T19:23:41Z : No. of bitstreams: 1 diniz_eg_dr_jabo.pdf: 1766438 bytes, checksum: b28fd4b1e87d5ab565efa2f5084bd69a (MD5) / Pouco se sabe sobre os eventos morfológicos que ocorrem durante o desenvolvimento pré-natal das gônadas nas raças zebuinas. O objetivo deste estudo foi descrever os eventos morfológicos relacionados ao desenvolvimento pré-natal da gônada, incluindo a sua formação, identificação de células germinativas primordiais, surgimento de oogônios, oócitos e folículos em embriões e fetos da raça Nelore. Oitenta e um embriões e fetos bovinos, com idade variando de 26 a 240 dias após fecundação, foram coletados em frigoríficos. A idade dos fetos foi estimada a partir de medidas tomadas no sentido crânio-caudal e aplicadas à fórmula proposta por Rexroad et. al. (1974). O sexo foi identificado a partir de observações macroscópicas e usando a técnica do PCR (Polymerase Chain Reaction) somente quando as diferenças sexuais morfológicas não foram evidentes. Para histologia, as gônadas foram fixadas em líquido de Bouin por 24 horas. Após processamento histológico, cortes de tecido de 5mm, foram corados com hematoxilina-eosina. Os resultados mostraram que a crista gonádica se formou a partir de 29 dias após fecundação. No 34º dia, células germinativas primordiais foram identificadas. As oogônias surgiram em grande quantidade entre 50 e 100 dias e seu número reduziu drasticamente, atingindo números finais aos 140 dias. Os folículos primordiais, folículos em crescimento e antrais apareceram em média aos 95, 140 e 180 dias, respectivamente. Oogônias e folículos primordiais, de forma diferente dos folículos em crescimento, apresentaram diferenças significativas no seu diâmetro nos vários períodos estudados. Folículos antrais mostraram diâmetro médio de 96,92 l 31,07mm aos 180 dias , chegando atingir médias de1331,43 l 567,43mm aos 240 dias... / Little is known about morphological events occurring during the prenatal development of gonads in the Zebu breeds. The objective of this study was to describe the morphologic events related to the prenatal development of the gonad, including its formation, identification of primordial germinative cells, appearance of oogonia, oocytes and follicles in Nelore breed embryos and fetuses. Eighty-one bovine embryos and fetuses, with age range from 26 to 240 days following fecundation, were gathered in a local slaughter-house. The age of fetuses was estimated from measures taken in the cranium-caudal direction and applied to the formula proposed by Rexroad et. al. (1974). The sex was identified from macroscopic observations and using PCR (Polymerase Chain Reaction) technique only when the morphologic sexual differences were not evident. For histology, gonads were fixed into Bouin fluid for 24 hours. Then, 5mm-tissue cuts were stained with hematoxylin-eosin. The results showed that the gonadal ridge was developed from 29 days following fecundation. At the 34th day, primordial germ cells were identified. Oogonia arose in great quantity between 50 and 100 days and its number reduced dramatically, attaining final numbers at 140 days. The primordial follicles, growing follicles and antral follicles appeared on the average at 95, 140 and 180 days, respectively. Oogonia and primordial follicles, in a different way from growing follicles, presented significant differences in its diameter in the several periods studied. Antral follicles showed 96.92 l 31.07mm in diameter at 180 days, achieving means of 1331.43 l 567.43 mm at 240 days. The statistical analysis showed a positive and highly significant correlation (P< 0.01), between the oogonia diameter and its nucleus, as well as between the primordial and growing follicles with its oocytes and respective nuclei... (Complete abstract, access undermentioned eletronic address)