O saco vitelino é uma das membranas extra-embrionárias que desempenha um papel importante para a sobrevivência inicial do embrião, atua como fonte de nutrição durante o período em que a placenta verdadeira ainda não está completamente formada. É uma provável fonte de células tronco, o qual abriga as primeiras células do sangue durante o desenvolvimento em mamíferos, os eritrócitos, os quais expressam fatores de transcrição que especificam estas células a seu destino hematopoiético. O objetivo deste trabalho foi caracterizar as células tronco hematopoéticas provenientes do saco vitelino de embriões bovinos, em diferentes fases gestacionais, sendo estes coletados em abatedouro local. Para descrição da análise macroscópica e cultivo celular das células do saco vitelino, os embriões bovinos foram divididos em grupos de idade gestacional: Grupo I (25 a 29 dias), Grupo II (30 a 34 dias), Grupo III (35 a 39 dias), Grupo IV (40 a 44 dias) e Grupo V (45 a 50 dias) em que permaneceram mais tempo em cultura e apresentaram a formação de aglomerados celulares, diferente dos grupos IV e V (40 a 45 dias) em que permaneceram poucos dias em cultura e não apresentaram aglomerados celulares. Esta divergência relaciona-se à idade gestacional (45 a 50 dias), período em que se inicia a regressão do saco vitelino. Em citometria de fluxo os grupos I, II e III (25 a 39 dias) obtiveram características semelhantes, alta expressão de marcadores hematopoéticos (CD34, CD90 e CD117). Para os grupos IV e V (40 a 50 dias) observa-se um declínio da expressão de CD34 e CD117 (marcadores hematopoéticos) e no grupo V houve um acréscimo da expressão de CD45 (marcador para leucócito) confirmando que estas células não estão mantendo-se indiferenciadas As células demonstraram ser resistentes a criopreservação, capazes de formar colônias em matriz de Metilcelulose, mostraram a formação de colônias após 14 dias em cultivo e a morfologia para células sanguíneas (linfócitos e monócitos) foi confirmada na citologia celular. Na expressão gênica obteve-se baixa expressão do gene GATA3, níveis diferentes de expressão entre os grupos para o marcador RUNX1 e ANXA5. Dessa forma, nossos achados mais significativos comprovaram o isolamento de células hematopoéticas a partir do saco vitelino de embriões bovinos, sugerindo que este é uma fonte laboriosa, porém viável e eficaz para a obtenção de células tronco para futuras aplicações na terapia celular e gênica. / The yolk sac is one of the extra-embryonic membranes which plays an important role in early embryonic survival and serves as source of nutrition during the period where in the placenta is not completely true formed. The yolk sac is a likely source of stem cells, which have first blood cells during development in mammals, the red blood cells, which express transcription factors that specify these hematopoietic cells to their destination. This study aimed to identify and characterize hematopoietic stem cells from the yolk sac of bovine embryos at different stages of pregnancy, which are collected at a local slaughterhouse. For a description of the macroscopic and cellular culture of yolk sac cells, are as follows bovine embryos were divided into groups of gestational age: Group I (25 to 29 days), Group II (30 to 34 days), Group III (35 to 39 days ), Group IV (40 to 44 days) and Group V (45 to 50 days) which stayed longer in culture and showed the formation of cell clusters, different from groups IV and V (40-45 days) in few that remained days in culture and showed no cell clumps. This divergence is related to gestational age (45 to 50 days), during which begins regression of the yolk sac. In flow cytometry groups I, II and III (25 to 39 days) had similar characteristics, high expression of hematopoietic markers (CD34, CD90 and CD117). For the groups IV and V (40 to 50 days) it is observed a decrease in expression of CD117 and CD34 (hematopoietic markers) and in group V were increased expression of CD45 (leukocyte marker), confirming that these cells are not keeping Undifferentiated cells are shown to be resistant to cryopreservation, capable of forming colonies in methylcellulose matrix showed the formation of colonies after 14 days in culture morphology and to blood cells (lymphocytes and monocytes) was confirmed by cytology cell. In gene expression was low GATA3 gene expression, different levels of expression between the groups for the marker and RUNX1 ANXA5. Our most significant findings confirmed the isolation and identification of hematopoietic cells from the bovine embryo yolk sac, therefore, it is feasible and an effective way of obtaining stem cells for future applications in cell therapy and gene.

Orientador: Eunice Oba / Resumo: A congelação de sêmen é uma ferramenta de grande importância na reprodução de animais domésticos, auxiliando na difusão do do material genético, preservando-o por tempo indeterminado, além do maior aproveitamento do uso de reprodutores com genética superior comprovada. Para que a congelação de sêmen seja eficiente e alcance resultados satisfatórios com a inseminação, utiliza-se no processo o emprego de diluentes, que tem como função proteger a célula contra o choque térmico e manter o espermatozoide viável até o momento da inseminação. O uso de frações de leite como meio diluidor tem se tornado muito conhecida e de grande importância no processo, usando, por exemplo, as micelas de caseína que conferem função de proteção da membrana plasmática e manutenção da viabilidade espermática. Levando em conta essas informações, este trabalho teve como objetivo avaliar a ação do caseinato de sódio nas características seminais pós descongelação do sêmen utilizando diluentes a base de gema de ovo (BB) e o mesmo diluente acrescido de caseinato de sódio 2% (BC).No experimento I, foram colhidos 3 ejaculados de 8 animais (n=24) por eletroejaculação, este sêmen foi divido em duas alíquotas, uma era diluída em meio comercial a base de gema de ovo e a outra alíquota diluída no mesmo meio mas acrescido de 2% de caseinato de sódio, em seguida foram envasadas em palhetas francesas com volume de 0,25 ml e refrigerado 4 horas á 5 °C em seguida congelado em nitrogênio líquido. Após a congelação as amo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The reproduction of domestic animals, helping in the diffusion of the genetic material, preserving it indefinitely, in addition to the greater use of reproducers with proven superior genetics. In order for semen freezing to be efficient and achieve satisfactory results with insemination, the use of diluents is used in the process, which has the function of protecting the cell against thermal shock and keeping the sperm viable until the time of insemination. The use of milk fractions as a diluting medium has become very well known and of great importance in the process, using, for example, the casein micelles that provide a protective function of the plasma membrane and maintenance of sperm viability. Taking this information into account, this study aimed to evaluate the action of sodium caseinate on semen characteristics after semen thawed using egg yolk (BB) diluents and the same diluent plus 2% sodium caseinate (BC) In experiment I, 3 ejaculates were collected from 8 animals (n = 24) by electroejaculation, this semen was divided into two aliquots, one was diluted in commercial medium based on egg yolk and the other diluted in the same medium but added of 2% sodium caseinate, then they were packaged in French straws with a volume of 0.25 ml and refrigerated 4 hours at 5 ° C then frozen in liquid nitrogen. After freezing, the samples were evaluated by computerized analysis of sperm movement (CASA), integrity of plasma and acrosomal membranes and generation of superoxide anion... (Complete abstract click electronic access below) / Mestre

In meeting the high demand for monoclonal antibodies, the chicken immunoglobulin system was exploited to generate recombinant antibodies against multiple target antigens. Following simultaneous immunisation of two chickens with a mixture of Plasmodium falciparum recombinant lactate dehydrogenase (LDH), histidine rich protein II (HRPII) and aldolase (ALDO), recombinant trypanosome variable surface glycoprotein (VSG) and malignant catarrhal fever virus (MCFV) each chicken produced egg yolk antibodies (IgY) against four of the five antigens. Using phage display technology, two single-chain variable fragment (scFv) antibody libraries, one with the immunoglobulin VH and VL chain regions joined by a single amino acid (G) and the other with a 15 amino acid flexible linker [(G4S) 3] were constructed using pooled splenic RNA. The single amino acid-linked scFv repertoire was evaluated as a source of highly specific diagnostic antibodies by panning against each of the five different antigens. After two rounds of panning, polyclonal phage ELISA showed the presence of antigen-specific phage antibodies against three (LDH, HRPII and VSG) of the five antigens. Five different anti-LDH and six different anti-HRPII scFvs were identified by sequence analysis. Evidence of high levels of antigen-driven gene conversion events was found in the framework and complementary determining regions and the VL chain pseudogene donors were identified. Stability of the selected scFvs was determined by incubation at different times and at different temperatures. The specificity and potential use of an LDH-specific scFv as a diagnostic reagent was shown in sandwich and competitive inhibition ELISAs. / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2007. / Veterinary Tropical Diseases / unrestricted

The amniote egg was a key innovation in vertebrate evolution because it supports an independent existence in terrestrial environments. The egg is provisioned with yolk, and development depends on the yolk sac for the mobilization of nutrients. We have examined the yolk sac of the corn snake Pantherophis guttatus by the dissection of living eggs. In contrast to the familiar fluid-filled sac of birds, the corn snake yolk sac invades the yolk mass to become a solid tissue. There is extensive proliferation of yolk-filled endodermal cells, which associate with a meshwork of blood vessels. These novel attributes of the yolk sac of corn snakes compared with birds suggest new pathways for the evolution of the amniote egg.

Reproductive mode has been remarkably labile among squamate reptiles and the evolutionary transition from oviparity to viviparity commonly has been accompanied by a shift in the pattern of embryonic nutrition. Structural specializations for placental transfer of nutrients during intrauterine gestation are highly diverse and many features of the extraembryonic membranes of viviparous species differ markedly from those of oviparous species. However, because of a high degree of evolutionary divergence between the species used for comparisons it is likely that the observed differences arose secondarily to the evolution of viviparity. We studied development of the extraembryonic membranes and placentation in the reproductively bimodal lizard Lacerta vivipara because the influence of reproductive mode on the structural/functional relationship between mothers and embryos can best be understood by studying the most recent evolutionary events. Lecithotrophic viviparity has evolved recently within this species and, although populations with different reproductive modes are allopatric, oviparous and viviparous forms interbreed in the laboratory and share many life history characteristics. In contrast to prior comparisons between oviparous and viviparous species, we found no differences in ontogeny or structure of the extraembryonic membranes between populations with different reproductive modes within L. vivipara. However, we did confirm conclusions from previous studies that the tertiary envelope of the egg, the eggshell, is much reduced in the viviparous population. These conclusions support a widely accepted model for the evolution of squamate placentation. We also found support for work published nearly 80 years ago that the pattern of development of the yolk sac of L. vivipara is unusual and that a function of a unique structure of squamate development, the yolk cleft, is hematopoiesis. The structure of the yolk sac splanchnopleure of L. vivipara is inconsistent with a commonly accepted model for amniote yolk sac function and we suggest that a long standing hypothesis that cells from the yolk cleft participate in yolk digestion requires further study.

The study includes separate papers, which are all linked by their emphasis on the effect of different diets on the impact of the development of the small intestine of the young ostrich. This abstract is intended to provide readers with a broad overview of the outcomes of the study. Part 1: Yolk utilisation and the development of the small intestines Chapter 2 deals with the composition of egg yolk as it is absorbed by starved ostrich Struthio camelus L. ) chicks from one to seven days post-hatching and for ostrich (Struthio camelus L.) chicks from one to sixteen days post-hatching on a pre-starter broiler diet. Chapter 3 provides information on the intestinal enzymes of ostrich (Struthio camelus L. ) chicks from one to sixteen days post-hatching on a pre-starter broiler diet. The effect of growth on enzyme activity immediately post-hatching up to sixteen days of age was studied. Chapter 4 details a histological and morphological study of the gastrointestinal tract of ostrich (Struthio camelus L.) chicks from two to sixteen days post-hatching on a pre-starter broiler diet. The effect of growth on histological and morphological changes in the various parts of the small intestines was examined. Part 2: Influence on various pre-starter diets on growth and the development of the intestinal tract Chapter 5 discussed a growth and digestibility study of ostrich (Struthio camelus L.) chicks on eight different pre-starter diets. The performance, growth and physiological development of the chicks on high and low variations of different nutritional components were examined. Chapter 6 provides information on certain intestinal enzymes of ostrich (Struthio camelus L.) chicks on the eight different pre-starter diets. Chapter 7 dealt with the histological and morphological changes of the gastrointestinal tract of ostrich (Struthio camelus L.) chicks as a result of high and low variations of different nutritional components in prestarter diets. Implications The first part of this study revealed that although it appeared that yolk content was absorbed faster in starved ostrich chicks, intake of external feed had a positive influence on the absorption of fat from the yolk. The changes in the fatty acid composition of the yolk fat content that were observed may indicate that ostrich chicks have the ability to withdraw certain fatty acid components, especially unsaturated fatty acids. The digestive tract of ostrich chicks was compared with that of broiler chicks and differences observed in amylase and lipase activity between the birds in this trial and in poultry could be due to genetic differences between species. It could also be due to the genetic homogeneity of the poultry chicks that were used in the trials, whereas ostrich chicks have not been subjected to the same extent of advanced breeding improvement to date. Results on histological and morphological growth of the small intestines of the ostrich chick indicate increased absorption with an increase in age. The second part of this study revealed that ostrich chicks performed differently on diets formulated with different nutritional components. It appears that a high fat and low sugar content in the diet is advantageous in ostrich pre-starter diets. Although there were a few mortalities during the trials, there is no clear indication as to why the chicks died, as the causes of mortalities were not investigated. Differences in composition of the eight pre-starter diets did not seem to have an influence on the protein content or enzyme activity of the small intestines of ostrich chicks. An anti-nutritional factor within certain feedstuffs may, however, have had an influence on enzyme activity. Similar results were obtained for the histological and morphological parameters which were measured in chicks fed the different diets. There was no single diet that promoted intestine development, although it seemed as if the small intestines of the chicks fed the high protein diet were under higher digestive stress than those of the chicks fed the other diets. / Thesis (PhD)--University of Pretoria, 2011. / Anatomy and Physiology / Unrestricted

Chick embryos derive nutrients from the yolk during incubation and transition to intestinal absorption posthatch. Nutrient uptake is mediated by digestive enzymes and membrane bound transporter proteins. The objective of this study was to determine expression profiles of nutrient transporters and digestive enzymes during incubation in the yolk sac membrane (YSM) and small intestine of Leghorn and Cobb chickens derived from 22–30 wk (young) and 45–50 wk (old) breeder flocks. Genes examined included peptide transporter PepT1, amino acid transporters EAAT3, CAT1, and B0AT, monosaccharide transporters SGLT1 and GLUT5, and digestive enzymes APN and SI. Expression of these genes was measured in YSM at embryonic day (e) 11, 13, 15, 17, 19, 20, and 21 and small intestine at e15, e17, e19, e20, and e21. Absolute quantification real-time PCR assessed gene expression. PepT1, APN, and B0AT expression in YSM peaked between e15 and e17 and then decreased until e21, while expression increased over time in the small intestine. SGLT1 and EAAT3 expression increased over time in the small intestine and YSM. There was minimal gene expression of SI in the YSM, while the small intestine had high expression. GLUT5 and CAT1 expression decreased in the YSM, while peaking at e19 then decreasing in the small intestine. Breed and flock age affected expression levels in some genes. These results demonstrate that these genes show tissue- and development-specific patterns of expression and that the YSM expresses many digestive enzymes and nutrient transporters associated with the small intestine. / Master of Science

Thyroid hormone content in eggs varies with the thyroid status of the hen and may influence embryonic development prior to the release of appreciable amounts of thyroid hormones by the embryonic thyroid gland. Methods for the measurement of thyroid hormones in egg yolk were verified by demonstrating consistency in the recovery of yolk thyroid hormones following a methanol/chloroform extraction, and in the measurement of thyroid hormones in extracts across a range of dilutions by RIA. Untreated hens produced eggs with yolk T₄ that was low relative to plasma T₄, but yolk T₃ that was comparable to plasma T₃. Hens dosed twice daily with L-thyroxine (T₄; 1x or 3x the daily thyroid secretion rate of T₄ per dose) showed significant increases in T₄ concentrations in their plasma and in the yolk of their eggs. Maternal thyroid hormone deposition in yolk varied with hens’ thyroid status. T₄ dosed hens demonstrated “levels” of hyperthyroidism and deposited greater amounts of T₄ into egg yolk compared to controls, yet appeared to regulate T₄ deposition into egg yolk at each level. Pelvic cartilage, a thyroid hormone responsive tissue, showed enhanced growth and differentiation in embryos from eggs of hens given the highest dose of T₄. Specifically, alkaline phosphatase activity (a marker of differentiation) and pelvic cartilage weights were significantly greater in embryos from high T₄ eggs than in controls. However, chicks from high T₄ eggs did not have measurable differences in total thyroid hormone content (carcass without the thyroid gland), general body growth (body weight, length, and general morphology), or hatchability when compared to controls. / Master of Science

Made available in DSpace on 2016-06-02T20:21:21Z (GMT). No. of bitstreams: 1 2667.pdf: 1236621 bytes, checksum: 9702ce62be0433e7639201366d6444aa (MD5) Previous issue date: 2009-06-30 / Financiadora de Estudos e Projetos / Species of the fraterculus group are associated to the biggest damages to fruit crops due to the fact that they attack indiscriminately green and ripe fruits, making them pests of great economic importance. These species are hard to tell apart, even showing some potential criptic species. The understanding of the populational structure and biology of this species group is of paramount importance to strategies of management and control of these pests. Here, we investigate variation on the gene yolk em 37 individuals sampled throughout the species distribution in Brazil seeking to better understand the populational structure of this species group in Brazil. Our data indicated existence of recombination, which lead us to analyze three different regions in the gene, 5 , mid, and 3 . These regions show high levels of nucleotide diversity intra and interspecifically for all three gene regions investigated. The use of Nested Clade Phylogenetic Analysis independently used on these regions indicate two main results that occurred more than one throughout our analyses. The first is a range expansion from north-NE populations towards the south, mostly related to specimens of Anastrepha fraterculus. The second common event, detected eight times, is restricted gene flow with isolation by distance (IBD). Dating of such events indicated that they are temporarily congruent which might indicate that the lack of IBD in other levels of the analysis might be caused either by sampling limitations or an excess of local gene flow that tampers out as we move farther in space. Our results of the gene yolk have provided us with a better understanding of the levels of local variation of this marker of for the species that may help us determine the evolutionary processes that shaped the species group current distribution. / Espécies do grupo fraterculus estão relacionadas com os maiores danos a culturas de frutas carnosas por atacar frutos verdes e maduros indistintamente, o que as torna pragas de grande importância econômica. Contudo, estas espécies são de difícil distinção, com a existência potencial de diversas espécies crípticas. Dessa forma, o entendimento da biologia e, particularmente, da estrutura populacional desses insetos-praga tem grande importância para o desenvolvimento de novas estratégias de manejo. Neste trabalho, investigamos a variação no gene yolk em 37 indivíduos ao longo da distribuição do grupo no Brasil para conhecermos melhor o padrão estrutural das espécies do grupo fraterculus. Os dados indicam a existência de recombinação, de forma que analisamos a região amplificada separadamente por regiões distintas: 5 , mid e 3 . Análises de polimorfismos nestas regiões apontaram para altos valores de diversidade nucleotídicas intra e interespecíficas para as três regiões gênicas em questão, sendo estes valores maiores para a região 3 . A metodologia da Análise dos Clados Aninhados (NCPA) foi utilizada para inferências de possíveis relações entre a configuração das redes haplotípicas com o padrão de distribuição geográfica considerando estas três regiões distintas. Através do uso desta metodologia, observamos dois eventos principais que podem estar influenciando a distribuição das espécies do grupo fraterculus no território nacional. A primeira inferência refere-se à expansão populacional no sentido Centro-Sul do Brasil, referente preferencialmente à espécie Anastrepha fraterculus. O segundo evento de inferência indica fluxo gênico restrito com isolamento por distância ao longo da distribuição global dos espécimes amostrados. A datação destes eventos indica uma congruência temporal, o que pode indicar que a não ocorrência de fluxo gênico em clados inferiores possa estar associado a restrições amostrais, uma vez que nossa amostragem é de fato restrita. Além disso, em populações mais próximas, o fluxo gênico pode estar ocorrendo com freqüência suficiente para promover a homogeneização genética das populações. Os resultados obtidos mediante a análise das seqüências do gene yolk nos permitiu um melhor conhecimento àcerca dos níveis de variação referentes a esse marcador, bem como determinar os processos que possam estar envolvidos no padrão de distribuição atual das espécies do grupo fraterculus no território nacional.

The main purpose of the project was to investigate existing markets within the food- and cosmetic industry in order to determine a strategy for market entrance with the bonus product of Fresenius Kabi. The original bonus product, P080, is a rest product from the process where phospholipids are extracted from egg yolk powder. P80 is a refine version of the P080 where egg yolk oil has been extracted, hence the protein content in P80 is higher. The extracted egg yolk oil is the final portion of the bonus product. Thereby, the original bonus product can be divided into two refined fractions. This project investigates the potential for each of these three substances to be a part of a fictive product.  By scanning the global protein market as well as the egg yolk protein and egg oil market, seven potential product segments were discovered. These segments were further evaluated in order to bring out specific products that were potential candidates as products based on the bonus product.  The analysis resulted in two fictive products based on P080; high value nutrition bars and snacks for seniors, one based on P80; protein powder, and one based on egg yolk oil; dietary supplement. All presented in three business cases.