Darbo tikslas – išanalizuoti skirtingų veislių karvių produktyvumo rodiklius ir somatinių ląstelių skaičiaus kaitą laktacijos eigoje. Uždaviniai: 1. Išanalizuoti ir palyginti pieno sudėties rodiklius tvartiniu ir ganykliniu laikotarpiais. 2. Išanalizuoti ir palyginti pieno kiekio kitimus skirtingais laikotarpiais. 3. Išanalizuoti ir įvertinti somatinių ląstelių skaičiaus kaitą. Tirtas X ūkyje Kėdainių rajone auginamų karvių produktyvumas ir SLS karvių piene. Tyrime naudotos 4 veislių antros-ketvirtos laktacijos karvės (po 15): Lietuvos juodmargės, holšteinai, Danijos juodmargės ir Lietuvos juodmargių x holšteinų mišrūnės. Išvados: Laikymo laikotarpis turėjo įtakos Lietuvos juodmargių ir holšteinų veislės karvių pieno primilžiams – tvartiniu laikotarpiu šių veislių karvės davė statistiškai didesnį vidutinį pieno kiekį per parą.Tvartiniu laikotarpiu Lietuvos juodmargių x holšteinų mišrūnių, Danijos juodmargių ir Lietuvos juodmargių veislių karvių pieno riebumas atitinkamai 0,41 proc., 0,42 proc. ir 0,49 proc. buvo didesni (p < 0,05) už holšteinų veislės karvių pieno riebumą. Vidutiniam baltymų ir laktozės kiekiui piene laikymo laikotarpis įtakos neturėjo – visų veislių karvių piene šių rodiklių reikšmės svyravo nežymiai: vidutinis baltymų kiekis – nuo 3,43 iki 3,73 proc., laktozės – nuo 4,27 iki 4,50 proc. Ganiava statistiškai reikšmingai padidino urėjos kiekį holšteinų (7,13 mg/100 ml), Lietuvos juodmargių (8,7 mg/100 ml), Lietuvos juodmargių x holšteinų mišrūnių (9,39... [toliau žr. visą tekstą] / Work objective – to analyse the rates of cows‘ productivity and milk quality according to the seasonality. Tasks: 1. To analyse season‘s influence on milk quantity provided by the different breeds of cows. 2. To analyse season‘s influence on milk composition and quality provided by the different breeds of cows. The research was performed on the cows‘ milk productivity and composition at the X farm in the district of Kedainiai. There were used cows of 4 breeds (15 cows) during theresearch: Lithuanian black and white cattle, holstein, Danish black and white cattle and crossbred Lithuanian black and white cattle and holsteins. The cows were of the third lactation. Conclusions: Seasonality had an influence on the milk yield of the Lithuanian black and white cattle and holsteins – during the indoor period the cows of these breeds gave statistically greater average milk quantity per twenty-four hours, respectively – 2,47 and 7,29 litres. During the indoor period cow milk fat of the breeds of crossbred Lithuanian black and white cattle and holsteins (0.41 % per unit), Danish black and white cattle (0.42 % per unit) and Lithuanian black and white cattle (0.49 % per unit) was statistically greater (p < 0,05). Seasonality did not have an influence on the average quantity of protein and lactose – the average values of these parameters in all breeds‘ cow milk ranged slightly: an average protein quantity – from 3.43 to 3.73 %, an average lactose quantity – from 4.27 to 4.50%. Pasturage... [to full text]

Darbo tikslas: Išanalizuoti ir įvertinti veiksnius, turinčius įtakos karvių mastitų sukėlėjų rūšims ir paplitimui Lietuvoje. Darbo uždaviniai: 1. Įvertinti ir aprašyti Lietuvoje paplitusius karvių mastito sukėlėjus; 2. Statistiškai įvertinti ir aprašyti veiksnių (metų, sezono ir apskrities) įtaką mastitų sukėlėjų rūšims ir paplitimui kontroliuojamų karvių piene 2011 metais; 3. Atlikti mastitų sukėlėjų jautrumo antibiotikams duomenų analizę ir statistikai įvertinti priklausomybę tarp antibiotikų. Magistro baigiamajame darbe buvo panaudoti VĮ „Pieno tyrimai“ laboratorijoje bakteriologiškai tirtų karvių, sergančių mastitu, duomenys. 2011metais buvo ištirta 2315 mastitu sergančios karvės. Dažniausiai identifikuota buvo Stphylococcus aureus - 33 proc., Staphylococcus spp - 21 proc.,gramneigiamos lazdelės – 13 proc.,Streptococcus spp- 11 proc., Streptococcus agalactiae – 11 proc. Duomenų analizė parodė, kad mastitų sukėlėjai didžiausia problema pieno kokybei tampa spalio, lapkričio ir gruodžio mėnesiais, t.y. tvartiniu laikotarpiu. Taip pat buvo nustatyta,kad mastitų sukėlėjai jautriausi buvo gentamicinui, amoksicilinui ir amoksicilino mišiniui su klovulanine rūgštimi.Analizė parodė, kad stipri teigiama koreliacija (r = 0,79) tarp mastito gydymui naudojamų antibiotikų buvo amoksicilino ir penicilino bei amoksicilino + klovulaninės rūgšties ir amoksicilino (r = 0,69). / The aim of work: Analyse and evaluate the factors, which affect cows mastitis pathogens in Lithuania. Work objectives: 1. Evaluate and describe widespread cow mastitis pathogens in Lithuania; 2. Statistically evaluate and describe the factors impact (of the year; of the season and of the country) on mastitis pathogens species and the spread in controlled cow milk; 3. Do the analysis of mastitis pathogens sensitivity to antibiotics and statistically evaluate addiction between antibiotics. In master thesis was used laboratory’s VĮ „Pieno tyrimai“data about cows with mastitis, which was bacteriological tested. In 2011 were investigated 2315 cows with mastitis. The most commonly identified Stphylococcus aureus – 33 percent, Staphylococcus spp - 21 percent, gram-negative bacteria – 13 percent, Streptococcus spp- 11 percent, Streptococcus agalactiae – 11 percent. Analysis of the data showed that the biggest problem of mastitis pathogens for milk quality becomes on October, November and December, during the stable period. It was also found that mastitis pathogens were sensitive to gentamicin, amoxicillin and amoxicillin mixture with clavulanic acid. Analysis had shown that a strong positive correlation (r = 0.79) between antibiotics for the treatment of mastitis were amoxicillin and penicillin also between amoxicillin + clavulanic acid and amoxicillin (r = 0.69).

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2006. / Additional advisors: Vithal K. Ghanta, Denise R. Shaw, Stephen A. Watts, Bradley K. Yoder. Description based on contents viewed Feb. 20, 2009; title from PDF t.p. Includes bibliographical references.

A fixed population of oocytes within primordial follicles, established prior to or just after birth has been firmly believed to support the postnatal mammalian ovary throughout an individual’s reproductive lifespan. However, the identification and isolation of cells from adult mammalian ovaries characterised by the expression of both germ and stem cell markers, suggest the presence of mitotically active cells, termed oogonial stem cells (OSCs) that may have the potential to produce new oocytes in the postnatal mammalian ovary. Putative OSCs have been isolated from adult tissues of several mammalian species, including rodents and humans. Upon reintroduction to the ovarian niche, human and rodent OSCs have generated new oocyte like structures which, at least in rodents, have generated functional oocytes capable of fertilisation and subsequent embryonic development to produce healthy offspring. We hypothesised that OSCs could be isolated from adult bovine ovaries and upon establishment within the appropriate ovarian niche could initiate successful oogenesis. To investigate this hypothesis, we have utilised fluorescently activated cell sorting (FACS) to isolate putative bovine OSCs (bOSCs) and an ovarian aggregate model, in vitro and in vivo to explore the oogenic potential of these cells. Putative bOSCs were successfully isolated by FACS based on the cell surface expression of germ cell marker DDX4 and established in culture. Pluripotency (LIN28 and OCT4) and germ (IFITM3, PRDM1, C-KIT and DAZL) cell associated genes were expressed in putative bOSCs established in culture. However, DDX4 transcripts were not consistently observed throughout bOSC culture. Aggregation of putative bOSCs with neonatal murine ovarian somatic cells to form chimeric ovarian aggregates, cultured in a hanging drop model for 7 days maintained germ cell phenotype, marked by DAZL expression. A subpopulation of putative bOSCs showed a spherical morphology, an increase in cell size and an association with neighbouring cells. Xenotransplantation of chimeric ovarian aggregates to the kidney capsule of immune deficient mice for 21 days generated multi-laminar follicles and structures with morphological similarities to primordial follicles (termed pre-primordial follicle-like structures). RNA Scope was unsuccessful in determining the origin of oocytes within chimeric ovarian aggregates. However, oocytes from pre-antral follicles in chimeric ovarian aggregates (n=6; 60.9± 3.6 μm, mean ± SEM) were significantly (P < 0.0001) larger than murine oocytes (n=38; 34.5± 1 μm, mean ± SEM) aggregated with murine ovarian somatic cells as positive controls, suggesting that these oocytes are undergoing different growth dynamics. This work has shown that putative bOSCs characterised by the expression of pluripotency and germ cell associated genes are present within adult bovine ovarian tissue and can be isolated using FACS and established in culture. These data also suggest that putative bOSCs may have the potential to undergo oogenesis and illustrate the potential use of these cells as a tool to investigate germ cell differentiation.

Made available in DSpace on 2014-06-11T19:27:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-07-29Bitstream added on 2014-06-13T19:15:06Z : No. of bitstreams: 1 serrao_ls_me_botfmvz.pdf: 217211 bytes, checksum: eb5988828907239da726996b3ad02487 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / Delineou-se o presente trabalho com o objetivo de avaliar os efeitos de três sistemas de manejo, com diferentes idades à desmama dos cordeiros (48horas, 30 e 45 dias) sobre a produção, composição do leite e incidência de mastite. Utilizaram-se 68 ovelhas Bergamácia distribuídas em três grupos por ordem de parição e idade. A dieta utilizada para os sistemas foi composta de silagem de sorgo e concentrado, na proporção volumoso: concentrado de (40:60), segundo exigência do NRC (1985). Os sistemas foram denominados de acordo com a idade à desmama dos cordeiros, sendo D48h = desmama às 48 horas após o parto e aleitamento artificial até 45 dias; D30d = desmama aos 30 dias e as ovelhas submetidas à ordenha somente após este período; D45d= desmama aos 45 dias com ordenha simultânea à amamentação. Neste último sistema os cordeiros permaneceram com suas mães em pasto durante 8 horas/dia e foram separados no final da tarde. As ovelhas dos três sistemas foram ordenhadas mecanicamente uma vez ao dia, às 8h 00, com produção de leite mensurada diariamente durante 90 dias para os sistemas D48h e D45d e 60 dias para o sistema D30d. Amostras de leite foram coletadas semanalmente para determinação da composição centesimal e contagem de células somáticas. A produção média total de leite das ovelhas do sistema D48h foi similar às ovelhas do sistema D45d e D30d (28,26; 28,65; 18,79 kg/ovelha/lactação, respectivamente). Nas três primeiras semanas, quando apenas os sistemas D48h e D45d foram avaliados, o teor médio de gordura, proteína e sólidos totais apresentaram-se superiores no D48h quando comparados ao D45d. A contagem de células somáticas foi similar entre os sistemas e apenas na primeira semana de lactação as ovelhas do sistema D45d apresentaram menor CCS em relação as ovelhas do sistema D48h. / This work aimed to evaluate the effects of three management systems, with different ages of lambs at weaning (48 hours, 30 and 45 days), on production, and composition of milk e incidence of mastitis. It was used 68 Bergamasca ewes allocated in three groups by order of parturition and age. The diet used for the systems was made up with sorghum silage and concentrate, in the volumous:concentrate ratio of (40:60), according to NRC (1985). Systems were named according to lambs’ age at weaning, being D48h = weaning 48 hours after parturition and artificial nursing up to 45 days; D30d = weaning at 30 days of age and sheep submitted to milking only after this period; D45d = weaning at 45 days of age with simultaneous milking. In this latter system, lambs remained with their mothers in pasture during 8 hours/day and were separated in the evening. Sheep from the three systems were mechanically milked once a day, at 8h 00 with milk production measured daily during 90 days for D48h and D45d and 60 days for D30d. Milk samples were collected weekly for determination of centesimal composition and somatic cells counting. The final average milk production D48h ewes were similar for systems D45d and D30d, (28.26, 28.65 and 18.79 kg/ewe/lactation, respectively. In the first three weeks, when only systems D48h and D45d were evaluated, the percentage of milk fat, protein, lactose and total solids were higher in D48h when compared to D45d. Somatic cells counting was similar among systems and at the 1-wk test day the D45d ewe was lower SCC than D48h ewe.

Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2015-08-07T14:17:51Z No. of bitstreams: 0 / Made available in DSpace on 2015-08-07T14:17:51Z (GMT). No. of bitstreams: 0 / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2016-12-08T16:24:23Z (GMT). No. of bitstreams: 1 PGCA15MA184.pdf: 964703 bytes, checksum: 9b63fdeda6cbc9b89c700c8427b99a28 (MD5) Previous issue date: 2015-08-28 / The cryopreservation of somatic cells has been highlighted not only in the preservation of germplasm, but also in the maintenance of genotypes used for the induction of pluripotency, development of strains and cloning by nuclear transfer. The study of the effects of controlled stress in somatic cells or gametes have shown that, in addition to providing increased survival under experimental conditions, it may influence other parameters like the growth pattern in culture. Bovine fibroblasts obtained by standard procedure from a cartilage explant were evenly distributed in four experimental groups as described below: fibroblasts in culture were subjected for 4 minutes to negative pressure 200, 500 and 800 mbar, immediately (PN0h) or 3 hours before (PN3h) freezing, performed in a standard solution (10% DMSO in D-MEM). After trypsinization, the cells were loaded in 0.25 mL straws in a concentration of 1 x 106 cells / mL, and total volume of 200 &#956;L. The straws were sealed and stabilized in a refrigerator (5-7 °C) for 30 minutes, and then exposed to nitrogen vapor (4 cm above the surface) for 5 minutes when were dipped therein. Thawing was performed in a water bath at 36 °C for 20 seconds. Fibroblasts not subjected to pressure were used as frozen (CC) and fresh (FC) controls. There was evaluated the post-thaw viability and subsequently the cell replication index, based on the population doubling time (PDT) performed each 24 h intervals, during 8 days. At each evaluation, the cells of one well of each group were trypsinized and the number of viable cells determined by hemocytometric chamber after staining with Trypan Blue. PDT was determined using the algorithm available online (http://www.doubling-time.com): TD = t x lg2/(lgNt lgN0). Data were submitted to ANOVA and T student test, with 5% significance level. The average cell survival of control group (89.8%) and PN500 0h (88.1%) were higher than all other groups. The PDT average time of all frozen cells was 33.2 h. The PDT time was similar in fresh (27.5 ± 0.35 h), frozen control (30.1 ± 2.3 h) and PN500 0h (32.4 ± 1.6 h) groups. The lowest PDT time was observed in the PN800 0h (21,9h) group. The freezing in plastic straws with 10% DMSO was adequate to cryopreserve bovine fibroblasts allowing high survival rates. It was not observed negative effects of submission to the negative pressure just before freezing, and the level of 200 and 500 mbar resulted in growth rates similar to the obtained with fresh fibroblasts. Although the negative pressure has not increased cryotolerance in bovine fibroblasts, it determined changes in the behavior of these cells during the post-thaw culture. New evaluations should be performed to assess cells subjected to negative pressure in the development of animal clones / A criopreservação de células somáticas vem se destacando não apenas como aliada na preservação de germoplasma, mas também na manutenção de genótipos utilizados para a indução de pluripotencia, formação de linhagens e clonagem por transferência nuclear. O estudo dos efeitos do estresse controlado em gametas ou células somáticas tem demonstrado que, além de proporcionar maior sobrevivência em condições experimentais, outros parâmetros podem ser influenciados, como por exemplo, o padrão de crescimento em cultivo. Para isto, fibroblastos bovinos de primeira passagem, obtidos por procedimento padrão de explantação de cartilagem, foram distribuídos uniformemente em quatro grupos experimentais conforme descrito a seguir: Fibroblastos em cultivo foram submetidos por 4 minutos à pressão negativa de 200, 500 e 800 mbar, imediatamente (PN0h) ou 3horas antes (PN3h) do congelamento, realizado em solução padrão (10% DMSO em D-MEM). Após a tripsinização, as células eram envasadas em uma concentração de 1 x 106 células/mL, em palhetas de 0,25 mL, num volume total de 200 &#956;L. As palhetas eram seladas e estabilizadas em geladeira (5-7 &#730;C) por 30 minutos, expostas ao vapor de nitrogênio (4 cm acima da superfície) por 5 minutos, e então mergulhadas no mesmo. O descongelamento era realizado em banho-maria a 36 °C por 20 segundos. Fibroblastos não submetidos à pressão foram utilizados como controles congelado (CC) e fresco (FC). Foram avaliados a viabilidade pós-congelamento, e posteriormente o índice de replicação, baseado no tempo de duplicação da população (PDT) celular, com intervalos de 24h e duração de 8 dias. A cada avaliação, um poço de cada grupo era tripsinizado sendo o número de células viáveis determinado em câmara hemocitométrica, após a coloração com Azul de Trypan. O PDT foi 10 12 determinado utilizando-se o algoritmo disponível online (http://www.doubling-time.com): TD = t x lg2/(lgNt lgN0). Os dados foram submetidos à ANOVA e teste T Student, com 5% de significância. A sobrevivência celular média dos grupos controle (89,8%) e PN500 0h (88,1%) foram superiores aos outros grupos. O tempo médio de PDT de todas as células congeladas foi 33,2h; o tempo de PDT foi semelhante nos grupos fresco (27,5 ± 0,35 h), controle congelado (30,1 ± 2,3 h) e PN500 0h (32,4 ± 1,6 h), o menor tempo foi observado no grupo PN800 0h (21,9 h). As palhetas plásticas se mostraram adequadas para o congelamento de fibroblastos bovinos, não sendo observados efeitos negativos da submissão à pressão negativa imediatamente antes do congelamento, sendo os valores de 200 e 500 mbar os que possibilitaram as melhores taxas de crescimento. Muito embora o emprego de pressão negativa não tenha aumentado a criotolerância de fibroblastos bovinos, determinou mudanças no comportamento destas células durante o cultivo pós-descongelamento. Novas avaliações devem ser realizadas para avaliar a qualidade das células submetidas à pressão negativa, no desenvolvimento de clones animais

Orientador: Antonio Nader Filho / Banca: Oswaldo Durival Rossi Júnior / Banca: Edmar Soares Nicolau / Resumo: O processo de adoção da refrigeração do leite na fazenda como prática para manter a qualidade inicial do leite tem ocorrido nos últimos anos no Brasil de forma rápida. A implementação e obrigatoriedade deste processo possibilitaram o uso de tanques comunitários por pequenos produtores, os quais são instalados em uma propriedade e recebe o leite das propriedades vizinhas, para que os mesmos pudessem permanecer na atividade. Neste trabalho foi avaliado as características físico-químicas, microbiológicas, celulares e detecção de resíduos de antibióticos, do leite de nove propriedades, assim como do leite de conjunto destas propriedades, entregues em um tanque comunitário localizado no município de Sacramento-MG. As amostras foram obtidas em dois dias consecutivos, uma vez que o produto contido no tanque era enviado ao estabelecimento industrial em dias alternados, portanto, a cada 48 horas, durante os meses de Abril a Julho de 2005. Os resultados verificados permitem concluir que o uso de tanque comunitário pode proporcionar a obtenção de leite com qualidade físico-química, microbiológica e celular, atendendo os limites propostos pela legislação vigente desde que obedecidas as normas de boas práticas de produção. / Abstract: The adoption process of cooling the milk on the farm as a practice for maintain the initial quality of the milk has been going on the latest years in Brazil, quickly. Both implementation and mandatoriness of this process enabled the use of communitary reservoirs which are expanded container in a propriety being fed witch milk proved by the therd party so that they were capable of keeping on doing the same activity. This work had the cellular, microbiological and physical-chemical characteristics and also the presence of residues of the antibiotics, from the milk originated from nine farms, as well as the mix milk these proprieties delivered into a comumunitary reservoirs located in the municipal county of Sacramento, State of Minas Gerais. The samples were collected into two consecutive days, as the product held in the container was conveyed to the industrial sector at alternate days, and as such, at each 48 hours, during the months of April through July 2005. The results obtained allowed to conclude that the utilization of a communitary reservoirs can offer the acquisition of physical-chemical based milk, microbiota and cellular, assisting the limits proposed by the effective legilstation, provided that the best practices of production are obeyed. / Mestre

The overarching objective of this thesis was the development of an improved expression cell line for recombinant proteins, in which a transgene of interest can be inserted into a highly active gene locus using recombinase mediated cassette exchange. Random integration of transgenic DNA is a common route to achieve transgene expression. However, randomly integrated transgenes are susceptible to gene silencing over time, and do not show stable expression for extended periods in culture. Furthermore, every new cell clone generated requires regulatory approval. The improvement of expression strategies may significantly reduce the time required to bring novel recombinant protein products to the market. In order to identify a suitable expression locus for the integration of transgene expression cassettes, total protein samples were derived from the production cell lines HEK293 and CHO. Highly expressed proteins were isolated via 2D PAGE, and identified via peptide mass fingerprinting. Their promoter regions were then validated to express a recombinant transgene in HEK293 cells. The long term stability of these promoter regions was also assessed. Direct gene targeting of the highly active gene loci may or may not be possible in typical producer cell lines. Targeting of a murine homologue to these highly expressed CHO/HEK293 loci may be more efficient in a murine stem cell line. The transfer of a modified allele from HM1 murine embryonic stem cells, into a somatic cell line (HC11) was demonstrated in this thesis. These validated methods were then explored for the generation of viable HM1-HEK293 and HM1-CHO fusion hybrids. For these experiments, a fluorescence based fusion assay was generated, validated and used for in-situ monitoring of the cell fusion process. The random integration of transgenic DNA into mammalian genomes typically results in a highly unpredictable integration architecture. RMCE at such loci would be inefficient. However, a highly efficient RMCE reaction at (rare) single copy transgene integrations, may be possible under the correct conditions. RMCE at randomly integrated loci could therefore be more beneficial (for transgene expression) than random integration alone. This thesis explores this concept with the use of a randomly integrated RMCE platform, and subsequent selection of cell lines post RMCE attempts at these loci CRISPR/Cas9 technology was also applied to a highly expressed locus in HEK293 cells. A framework for successful direction of double strand breaks to a defined locus is demonstrated in this work. The methods used to achieve this can therefore be built upon for the homologous recombination of a transgenic cassette, into a highly expressed locus in HEK293 cells. Monoclonal antibodies have dominated the biologics market for over two decades, and mammalian expression systems are well suited to their production. The work in this thesis attempts to raise and verify antibody molecules against a potential tumour marker using hybridoma and phage display technologies.