A longevidade é uma característica relacionada à lucratividade da atividade leiteira. Contudo, sua seleção em rebanhos de vacas Holandesas no Brasil ainda é pouco considerada. Objetivou-se determinar os fatores não genéticos que influenciam a longevidade funcional em vacas Holandesas no Brasil, bem como estimar os parâmetros genéticos e conhecer a contribuição das características de tipo e da contagem de células somáticas (CCS) sobre o risco relativo de descarte das vacas. Utilizou-se um modelo de riscos proporcionais Weibull estratificado. Os efeitos fixos foram independentes do tempo, como a idade ao primeiro parto, e dependentes do tempo, como o efeito da região por ano de parto, classes de produção de leite por ano de parto dentro de rebanho, classes de percentagem de proteína e gordura dentro de rebanho, classes de produção de leite por número de lactações dentro de rebanho e variação nas classes de tamanho de rebanho. Os efeitos aleatórios foram: rebanho-ano, touro e de touro-avô materno. O risco de descarte aumentou com a idade ao primeiro parto, com o tamanho do rebanho, com o número de lactações e com o estágio de lactação. A produção de leite apresentou maior efeito sobre o risco de descarte. Vacas de baixa produção de leite, gordura e proteína apresentaram maior probabilidade de descarte em comparação à classe mediana. Vacas pertencentes às regiões do Paraná e São Paulo permaneceram mais tempo no rebanho do que as vacas de outras regiões. Os valores de h² variam de 7,8% a 6,1% para a h² equivalente e a efetiva, respectivamente. Observou-se tendência genética positiva para a longevidade. As características de tipo, escore final, angularidade, nivelamento da linha superior, textura do úbere e ligamento suspensório foram as características que se apresentaram mais relacionadas com a longevidade funcional. Foram observadas diferenças no risco de descarte dependendo do número de vacas classificadas para tipo dentro de rebanho. Até a 4ª lactação, o risco de descarte foi menor para vacas com baixa CCS em comparação a vacas da classe mediana. Já para vacas na 5ª lactação, a alta CCS conduziu ao menor risco de descarte. A rotina de avaliação genética é necessária para melhorar a duração da vida produtiva de vacas da raça Holandesa no Brasil. Características preditivas, tais como escore final, angularidade, nivelamento da linha superior, textura do úbere, ligamento suspensório e a CCS podem ser utilizadas para aumentar a confiabilidade dos valores genéticos dos touros para longevidade funcional. / Longevity is a trait related to the profitability of dairy activity. However, its selection in Brazilian Holstein herds is still little considered. The aim of this study was to determine the non-genetic factors that influence functional longevity in Holstein cows in Brazil, as well as to estimate the genetic parameters and the contribution of somatic cell score (SCS) and type traits on the relative culling risk of cows. A piecewise Weibullproportional hazard model was used. The fixed effects were time-independent, as age at first calving, and time-dependent, as the interaction effects of region by year of calving, milk production class by year of calving within herd, within herd milk production class by lactation number, within herd fat and protein content, and variation in herd size class. The random effects were herd-year effect, additive genetic contribution from the sire and maternal grandsire of the cow. The relative risk increased with age at first calving, lactation number by stage of lactation, and herd size but lower risks were observed when herd size was increasing or decreasing, compared to stable herds. Milk production had a greater effect on the risk of culling. The relative risk increased as milk production, protein and fat decreased, but to a lesser extent for protein and fat compared to milk yield. Cows from Paraná and São Paulo regions remained longer in the herd than cows from the other regions. The h² values varied from 7.8% to 6.1% for equivalent and effective h², respectively. A positive genetic trend of functional longevity was observed. The type traits, final score, angularity, top line, udder texture and suspensory ligament showed the strongest relationship with productive life. Differences in risk of culling were observed depending on the fraction of type-scored animals within a herd. The absence of type trait phenotypes was associated with a strong increase of culling risk for the cows. The impact of SCS on longevity was high in cows from 1st to 4th lactation with high SCS. Interestingly, for 5th lactation, cows with lower SCS have higher culling risk compared to cows with higher SCS. A routine of genetic evaluation is necessary to improve length of productive life of Brazilian Holsteins under local conditions. The use of early predictors correlated with longevity, as final score, angularity, top line, udder texture, suspensory ligament and SCS, may be recommended to increase the reliability of sires’ estimated breeding values for functional longevity.

The objective of my study was to analyse the factors negatively influencing a somatic cell count (SCC) in raw cow´s milk. Milk samples were tested in three breeds with different technology of breeding and milking for a time period of three years. The SCC values were determined by Fluoro-opto-electronic method on a Fossomatic apparatus. Significant factor influencing SCC was quality practise hygiene of mammary gland. The lowest average values of SCC were determined in breed where was practise thoroughly hygiene of mammary gland (avarage SCC in year 2006 was 223,3.103 SB.ml-1). Next factors negatively influencing SCC were age of cows, annual period, quality of feed and frequency exchanges a udder sleeve. Influence a technology of breeding, milking and size breed on somatic cell count in milk was not extended.

Graduation theses are inquired into question of somatic cell count (SCC) in row cow milk in relation to the used methods of mastitis prevention. In the year 2005 and 2006 was following values of SCC in raw cow milk in tetra breeding dairy cows. Breeds are differentating from each other with technology of breeding (horsy, summer pasture), technology of lairege and milking. SCC in bulk tank milk was determined according to ČSN EN ISO 133366 {--} 3 milk. Lowest average funds PSB (156,3 {$\cdot$} 10{$^3$} {$\cdot$} ml&#713;{$^1$}) was determined in breeding with summer pasture (loos pen bedding housing). Breed with stanchion bedded housing without pasture (SCC 277,3 {$\cdot$} 10{$^3$} {$\cdot$} ml&#713;{$^1$}) and breed with bedding{--}free slatted-floor housing without pasture (SCC 277,4 {$\cdot$}10{$^3$} {$\cdot$} ml&#713;{$^1$}) was in a tight spot with inadequancy hygiene of stable and milking. The highest average values of SCC were determinated in loose bedded cubicle housing without pasture (289,1 {$\cdot$} 10{$^3$} {$\cdot$} ml&#713;{$^1$}), where was deficiencies in feeding (feeding mouldy silage) and higher dustiness environment. We proved a statistically highly significant difference (P<0,001) between farm using summer pasture and farms without pasture. Main deficiencies in methods of mastitis prevention is that breeds doesn't use individual disposable tissue cloths for the udder wiping, farms doesn't use preddiping, inadequancy in hygiene of lairege and milking, feeding mouldy silage and inadequancy in ransack confirmed dairy cows. Key words: raw cow milk, somatic cell count, technology

Orientadores: Mirna Lucia Gigante, Marcos Veiga dos Santos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-05T00:26:38Z (GMT). No. of bitstreams: 1 Mazal_Guillaume_M.pdf: 1093019 bytes, checksum: a2931e0c265f17d65f21671d7680bb5a (MD5) Previous issue date: 2005 / Mestrado / Mestre em Tecnologia de Alimentos

Os objetivos gerais deste estudo foram avaliar o efeito suplementação de zinco (Zn), cobre (Cu) e selênio (Se) orgânicos para vacas leiteiras e os seus efeitos sobre a qualidade do leite, saúde da glândula mamária e consumo de alimentos. Os objetivos específicos foram avaliar: a contagem de células somáticas (CCS), a prevalência de mastite clínica e subclínica,a produção e composição de leite nos 80 primeiros dias de lactação; monitorar a atividade enzimática de superóxido dismutase (CuZnSOD), glutationa peroxidase (GSH-Px) e ceruloplasmina (CP); o consumo de alimentos; a concentração plasmática de Zn, Cu e Se; e as variações de peso e escore dos animais. Dezenove vacas leiteiras, com prenhez confirmada, foram selecionadas por peso, escore de condição corporal (ECC), número de lactações e produção de leite da lactação anterior, e distribuídas ao acaso em dois grupos para receber fontes de Zn, Cu e Se orgânica (n=9) ou inorgânica (n=10). As dietas foram formuladas para suprir os requerimentos nutricionais dos animais dos 60 dias antes da data prevista do parto aos 80 dias de lactação. Amostras dos alimentos fornecidos e das sobras foram coletadas diariamente para posterior análise de composição. O leite foi coletado semanalmente a partir da 3ª semana de lactação para determinação da composição e CCS, e nos dias 1 e 7 de lactação, e quando diagnosticados casos clínicos de mastite, para cultura microbiológica. Amostras de sangue foram coletadas aos -60, -21, 1, 21, 40 e 80 dias do período experimental para análises da concentração de CuZnSOD, GSH-Px e CP. Para determinação das concentrações plasmáticas de Zn, Cu e Se amostras de sangue foram coletas aos 60 dias antes da data prevista de parto, e no 1º, 40º e 80º dias de lactação. Avaliações do escore de condição corporal (ECC) e do peso corporal (PC) foram realizadas no início e final do experimento, no parto, e uma vez por semana durante todo o período experimental. A incidência (novos casos)e o total de casos de mastite subclínica foi menor para o grupo de vacas alimentadas com fontes orgânicas de Zn, Cu e Se em comparação com os animais que receberam fontes inorgânicas. A CCS durante os primeiros 80 dias de lactação foi menor (P = 0,056) para o grupo alimentado com Zn, Cu e Se orgânicos. Não foram observados efeitos de fontes orgânicas de Zn, Cu e Se sobre as concentrações de CuZnSOD , GSH-Px e CP, Zn, Cu e Se plasmáticos, produção e composição de leite, consumo de nutrientes, ECC, mudança de ECC e PC. Foi observado efeito (P=0,024) da fonte sobre a mudança de PC. / The general objectives of this study were to evaluate the effect organic zinc (Zn), copper (Cu) and selenium (Se) supplementationto dairy cows on milk quality, mammary gland health and feed intake. The specific objectives were to evaluate: the somatic cell count (SCC), clinical and subclinical mastitis prevalence, milk production and composition during the first 80 days of lactation; the superoxide dismutase (CuZnSOD), glutathione peroxidase (GSH-Px) and ceruloplasmin (CP) enzyme activity; the nutrients intake; Zn, Cu and Se plasmatic concentrations; changes in weight and body condition score. Nineteen dairy cows, with confirmed pregnancy, were selected by body weight (BW), body condition score (BCS), number of lactation, and milk yield in previous lactation, and randomly distributed among two groups to receive organic (n=9) or inorganic (n=10) sources of Zn, Cu and Se. The diets were formulated to meet the nutritional requirements of animals from 60 days before the expected date of calving up to 80 days of lactation. Every day, food samples and leftovers were collected for composition analysis. Milk samples was collected weekly after 15 days of lactation to determine the composition and CCS, on days 1 and 7 of lactation, and when a mastitis clinical case was diagnosed for microbiological culture. Blood samples were collected on -60, -21, 1, 21, 40 and 80 days of the experimental period for CuZnSOD, GSH-Px, and CP analysis. For plasma concentrations of Zn, Cu and Se blood samples were collected at 60 days before calving, and at 1st, 40th and 80th days of lactation. Assessments of body condition score (BCS) and body weight (BW) were performed at the beginning and at the end of the experiment, the day of calving, and once a week throughout the experimental period. The incidence (new cases) and total number of subclinical mastitis cases was lower for the group of cows fed organic Zn, Cu and Se in comparison with animals that received the inorganic sources. The SCC during the first 80 days of lactation was lower (P = 0,056) for the group fed organic Zn, Cu and Se. There were no effects of Zn, Cu and Se organic supply on concentrations of CuZnSOD, GSH-Px and CP, Zn, Cu and Se plasma, production and composition of milk, consumption of nutrients, BW, BCS and changes on BCS. It was observed effect of source on BW changes (P=0,024).

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-08-23T19:32:17Z No. of bitstreams: 1 naiaraaparecidarodriguesdeoliveira.pdf: 913286 bytes, checksum: a8b96b3b7d78237e6a66fbd73d141644 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-08-28T12:27:07Z (GMT) No. of bitstreams: 1 naiaraaparecidarodriguesdeoliveira.pdf: 913286 bytes, checksum: a8b96b3b7d78237e6a66fbd73d141644 (MD5) / Made available in DSpace on 2018-08-28T12:27:07Z (GMT). No. of bitstreams: 1 naiaraaparecidarodriguesdeoliveira.pdf: 913286 bytes, checksum: a8b96b3b7d78237e6a66fbd73d141644 (MD5) Previous issue date: 2018-02-28 / A mastite está entre as doenças do gado leiteiro que mais causam prejuízos em todo mundo. Entre os agentes causadores estão os patógenos de origem contagiosa Staphylococcus aureus e Streptococcus agalactiae que mais contribuem para o aumento da Contagem de Células Somáticas (CCS) em rebanhos infectados.Desde a implantação da Instrução Normativa 51 ( IN 51/2002) do Ministério da Agriculutura Pecuária e Abastecimento(MAPA) em 2005, pouco se avançou em melhorias no parâmetro CCS dos rebanhos brasileiros, fazendo-se necessário o levantamento da situação das regiões a fim de traçar estratégias específicas para o controle da mastite e atendimento aos requisitos da legislação. O objetivo do presente estudo foi estabelecer uma classificação baseada em risco (dados probabilísticos) (risco) para presença de S. aureus e S. agalactiae em rebanhos bovinos leiteiros localizados Zona da Mata do Estado de Minas Gerais e vinculados a uma cooperativa. O trabalho foi realizado utilizando a população de bovinos leiteiros vinculados à Cooperativa dos Produtores de Leite de Leopoldina de Responsabilidade Ltda (LAC). A avaliação da dependência espacial entre as coordenadas geográficas dos rebanhos e a média geométrica anual da contagem de células somáticas foi realizada por meio de semivariogramas e os mapas de isolinhas por meio do método de Krigagem. Amostras para análise de CCS foram coletadas mensalmente.Para o estudo de prevalência foram selecionados aleatoriamente 43 rebanhos dos quais foram coletadas 3 amostras consecutivas de cada rebanho para análises microbiológicas durante o período de junho de 2016 a novembro de 2017. A análise espacial apresentou grau de dependência espacial e coeficiente de determinação fracos (GD<0,25; r2<0,13). A distribuição de frequência da CCS mostrou que apenas 23,3% dos rebanhos atendem ao limite de 400.000 células/mL . A prevalência de S. aureus foi de 93% a de S. agalactiae de 81,4% não diferindo estatisticamente entre si. Dos 43 rebanhos analisados, em tres (7%) não houve isolamento de S.aureus e de S. agalactiae, cinco (11,6%) apresentaram isolamento somente de S. aureus e 35 (81%) apresentaram isolamento de ambos patógenos. Não houve rebanhos com isolamento somente de S. agalactiae. Dos rebanhos sem isolamento destes agentes, a média de CCS foi de 224.000 células/mL, com isolamento somente de S. aureus a média de CCS foi de 447.000 células/mL, e para rebanhos com isolamento dos dois patógenos, S. aureus e S. agalactiae, 794.000 células/mL. Os rebanhos sem isolamento ou somente com isolamento de S. aureus estão associados a CCS inferior a 400.000 células/mL e rebanhos com isolamento de ambos os patógenos estão associados a CCS superior a 400.000 células/mL. A utilização da curva ROC para dados de CCS de rebanhos associados ao estudo de prevalência de S. aureus e de S. agalactiae permitiu classificar rebanhos em tres níveis de risco: baixo, médio e alto. A prevalência de S. aureus e de S. agalactiae entre rebanhos foi considerada alta para ambos patógenos, indicando que as medidas de controle para estes patógenos não estão sendo realizadas de maneira eficiente. Não foi observada diferença entre as prevalências de S. aureus e de S. agalactiae entre os rebanhos. Foi observada uma associação entre a CCS dos rebanhos e a presença de ambos os patógenos, permitindo desta forma uma classificação dos rebanhos. / Mastitis is among the most damaging diseases in dairy cattle worldwide. Among the causative agents are the pathogens of contagious origin Staphylococcus aureus and Streptococcus agalactiae that contribute most to the increase of Somatic Cell Count (SCC) in infected herds. Since the implantation of IN 51 in 2005, little progress was made in improving the SCC in the Brazilian dairy herds, therefore it is necessary to survey the mastitis situation of the regions in order to devise specific control strategies for mastitis control and compliance with the legislation requirements . The objective of the present study was to establish a risk - based classification (risk) for the presence of S. aureus and S. agalactiae in dairy herds located in the Zona da Mata of the State of Minas Gerais associated to a cooperative. The work was carried out using the population of dairy herds of Cooperativa dos Produtores de Leite de Leopoldina de Responsabilidade Ltda. (LAC).The evaluation of the spatial dependence between the geographical coordinates of the herds and the annual geometric mean of the somatic cell count was performed using semi-variograms and isoline maps using the Kriging method. Milk samples for SCC analyses were collected monthly. For the prevalence study, 43 herds were randomly selected and three milk samples from each herd were collected for microbiological analyzes during the period of June 2016 to November 2017. The spatial analysis presented degree of spatial dependence and weak determination coefficient (GD <0.25; r2 <0.13) The frequency distribution of CCS showed that only 23.3% of the herds met the limit of 400,000 cells / mL. The prevalence of S. aureus (93%) and of S. agalactiae (81.4%) did not differ statistically from one another. Of the 43 herds analyzed, three (7%) was no isolation of S. aureus and S. agalactiae, five (11.6%) had isolation only of S. aureus and 35 (81%) presented isolation of both pathogens. There were no herds with isolation of S. agalactiae. The mean SCC was 224,000 cels / mL, for the herds with absence of S. aureus and S. agalactiae, and 794.000 cels/ mL when the this two pathogens were present. The mean SCC for the herds with isolation only of S. aureus was 447,000 cells / mL,. The abcense or isolation only S. aureus herds are associated with CCS less than 400,000 cells / mL and herds with isolation of both pathogens associated with CCS greater than 400,000 cells / mL. The use of the ROC curve for herd CCS data associated to the study of S. aureus and S. agalactiae prevalence allowed the classification of the herds at three levels of risk: low, medium and high. The prevalence of S. aureus and S. agalactiae among herds was considered high for both pathogens, indicating that the control measures for these pathogens are not being carried out efficiently. There was no difference between the prevalences of S. aureus and S. agalactiae among the herds. An association was observed between the SCC of the herds and the presence of both pathogens, thus allowing a classification of the herds.

The repetitive sequence composes nearly half of human and mouse genome, most of which are scattered repeats of transposable elements (TEs). The non-LTR retrotransposons are the most accumulated TEs in the mammalian genome and L1s are the most active and abundant autonomous retrotransposons. L1s are highly activated during the epigenetic reprogramming of early mammalian embryos and have the highest level of expression among all retrotransposons throughout the preimplantation state. Moreover, the reprogramming of somatic cells into iPSCs is associated with an increase in L1 expression. The transcription of L1 during the early embryogenesis is necessary to regulate developmental genes and prevent heterochromatin formation to maintain cellular pluripotency state, that guarantying an appropriate future differentiation. However, the role of L1 reactivation during the somatic cell reprogramming remains unclear. Therefore, aim of this work is to study the impact of L1 transcription during the reprogramming process of the iPSCs. We used CRISPR-mediated gene activation (CRISPRa) system that fuse a deactivated Cas9 (dCas9) with transactivation domains (VPR). We confirm the ability to overexpress L1 in Human Embryonic Kidney cells (HEK293) and Human Dermal Fibroblasts (HDFs) by utilizing CRISPR activation system and this will provide a good opportunity to study the role of L1 transcripts during the reprogramming of HDFs into iPSCs. Furthermore, we established stable HDFs that able to express combinations of “Yamanaka” reprogramming factors. The model system will allow to investigate the effect of overexpressing L1 with reprogramming factors to answer the question of whether L1 can trigger or facilitate the reprogramming processes and its underlying mechanism.

Research to determine the impact of PTA<SUB>SCS</SUB> on incidence of mastitis, daughter response to infection, and other measures of daughter performance was conducted using data on 1st, 2nd, and 3rd lactation Holsteins obtained from the Virginia Tech herd and from VA DHI herds. Overall correlation of PTA<SUB>SCS</SUB> to lactation average SCS ranged from 0.13 to 0.17 across all data sets. Correlations between PTA<SUB>SCS</SUB> and 1st lactation SCS measures were higher than those between PTA<SUB>SCS</SUB> and SCS in later parities, but higher correlations were found between 2nd and 3rd lactation SCS measures than between 1st and later parities. Correlation of lactation average SCS and incidence of clinical mastitis was 0.41. Regression of lactation average SCS and averages of test day SCS measures on PTA<SUB>SCS</SUB> was significant in 1st, 2nd, and 3rd lactations. All significant relationships were linear and equal or close to 1.0. Relationships between PTA<SUB>SCS</SUB> and number of cases of clinical mastitis (.79), number of treatments (2.0), number of days treated (7.0), changes in SCS from beginning to end of a lactation (-.26), and the slope of changes in test day SCS with DIM (5.9x10-4) were significant only in 1st lactation. No significant relationships between PTA<SUB>SCS</SUB> and measures of clinical mastitis or variation in test day SCS measures were found in 2nd or 3rd lactations. Heavy cull rates imposed on 1st lactation cows in the Virginia Tech herd explained lack of significance in the later parities in the herd study, but results in following analyses indicated that measures of SCS in 1st and later parities may be two different, but correlated, traits. The greatest impact of PTA<SUB>SCS</SUB> on measures of daughter performance and profit was the negative relationship between PTA<SUB>SCS</SUB> and herd life. Increased PTA<SUB>SCS</SUB> resulted in the decreased ability to survive involuntary culling, and thus decreased opportunity for lifetime yield and profit. Selection on PTA<SUB>SCS</SUB> should be an effective method of reducing incidence of clinical mastitis, lactation average SCS, and variation in SCS, or response to infection. The response, however, may be different in 1st lactation than in later parities. / Master of Science

The cells from the inner cell mass (ICM) of an early embryo have the potential to differentiate into all the different cell types present in an adult organism. Cells from the ICM can be isolated and cultured in vitro, becoming embryonic stem cells (ESCs). ESCs have several properties that make them unique: they are unspecialized, can self-renew indefinitely in culture, and given the appropriate cues can differentiate into cells from all three germ layers (ecto-, meso-, and endoderm), including the germline, both in vivo and in vitro. Induced pluripotent stem cells (iPSCs) can be generated from adult, terminally differentiated somatic cells by transient exogenous expression of four transcription factors (Oct4, Sox2, Klf4, and cMyc; OSKM) present normally in ESCs. It has been shown that iPSCs are equivalent to ESCs in terms of morphology, gene expression, epigenetic signatures, in vitro proliferation capacity, and in vitro and in vivo differentiation potential. However, unlike ESCs, iPSCs can be obtained from a specific individual without the need for embryos. This makes them a promising source of pluripotent cells for regenerative medicine, tissue engineering, drug discovery, and disease modelling; additionally, in livestock species such as the bovine, they also have applications in genetic selection, production of transgenic animals for agricultural and biomedical purposes, and species conservancy. Nevertheless, ESC and iPSC lines that meet all pluripotency criteria have, to date, only been successfully produced in mice, rats, humans, and non-human primates. In the first part of this dissertation, we attempted reprogramming of three types of bovine somatic cells: fetal fibroblasts (bFFs), adult fibroblasts (bAFs), and bone marrow-derived mesenchymal stem cells (bMSCs), using six different culture conditions adapted from recent work in mice and humans. Using basic mouse reprogramming conditions, we did not succeed in inducing formation of ESC-like colonies in bovine somatic cells. The combination of 2i/LIF plus ALK5 inhibitor II and ascorbic acid, induced formation of colony-like structures with flat morphology, that occasionally produced trophoblast-like structures. These trophoblast-like vesicles did not appear when an inhibitor of Rho-associated, coiled-coil containing protein kinase 1 (ROCK) was included in the medium. We screened for expression of exogenous OSKM vector with RT-PCR and found upregulation of OSKM vector 24h after Dox was added to the medium; however, expression was sharply decreased on day 2 after Dox induction, and was not detectable after day 3. In a separate experiment, we induced reprogramming of bFF and bAFs using medium supplemented with 50% of medium conditioned by co-culture with the bovine trophoblast CT1 line. These cells expressed both OCT4 and the OSKM vector 24h after Dox induction. However, similar to our previous observations, both markers decreased expression until no signal was detected after day 3. In summary, we were unable to produce fully reprogrammed bovine iPSCs using mouse and human protocols, and the exact cause of our lack of success is unclear. It is possible that a different method of transgene expression could play a role in reprogramming. However, these ideas would be driven by a rather empirical reasoning, extrapolating findings from other species, and not contributing in our understanding of the particular differences of pluripotecy in ungulates. Our inability to produce bovine iPSCs, combined with the only partial reprogramming observed by others, justifies the need for in depth study of bovine pluripotency mechanisms, before meaningful attempts to reprogram bovine somatic cells to plutipotency are made. Therefore, we focused on getting a better understanding of bovine nuclear reprogramming. This would allow us to rationally target the specific requirements of potential bovine pluripotent cells. Cell fusion is a process that involves fusion of the membrane of two or more cells to form a multinucleated cell. Fusion of a somatic cell to an ESC is known to induce expression of pluripotency markers in the somatic nucleus. In the second part of this dissertation, we hypothesized that fusion of bFFs to mouse ESCs (mESCs) would induce expression of pluripotency markers in the bFF nucleus. We first optimized a cell fusion protocol based on the use of polyethylene glycol (PEG), and obtained up to 11.02% of multinucleated cells in bFFs. Next, we established a method to specifically select for multinucleated cells originated from the fusion of mESCs with bFFs (heterokaryons), using indirect immunofluorescence. With this in place, flow cytometry was used to select 200 heterokaryons which were further analyzed using RNA-seq. We found changes in bovine gene expression patterns between bFFs and heterokaryons obtained 24h after fusion. Focusing on the bovine transcriptome, heterokaryons presented upregulation of early pluripotency markers OCT4 and KLF4, as well as hypoxia response genes, contrasted with downregulation of cell cycle inhibitors such as SST. The cytokine IL6, known to increase survival of early embryos in vitro, was upregulated in heterokaryons, although its role and mechanism of action is still unclear. This indicates that the heterokaryon cell fusion model recapitulates several of the events of early reprogramming, and can therefore be used for further study of pluripotency in the bovine. The cell fusion model presented here can be used as a tool to characterize early changes in bovine somatic nuclear reprogramming, and to study the effect of different reprogramming conditions on the bovine transcriptome. / Ph. D. / The cells of an early embryo have the potential to give rise to any cell type found in the adult body. When these cells are transferred to a culture dish and kept under the right conditions, they become Embryonic Stem Cells (ESCs), and they retain the same developmental potential as the original embryonic cells they were derived from. In 2006, researchers in Japan found that it is possible to “reprogram” the cells of an adult individual (for example, fibroblast skin cells taken from a biopsy) to an embryonic state, by forcing the cells to express extra copies of genes that are normally active in embryos. These reprogrammed cells are called induced Pluripotent Stem Cells (iPSCs), and similarly to ESCs, they also have the potential to produce any cell type found in an adult organism. Lines of iPSCs from livestock species have possible applications in agriculture, species conservancy, biomedical industry, and veterinary and human health. Unfortunately, for reasons that are to date not fully understood, the technology to produce iPSCs has, so far, only worked in mice, rats, humans, and non-human primates. We first attempted to produce bovine iPSCs by adapting methods and conditions used to derive iPSCs in mice and humans. We observed partial reprogramming of bovine cells, but were ultimately not able to produce true bovine iPSCs. This suggests that the bovine requires alternative/additional factors to induce reprogramming in adult cells. However, not knowing exactly what conditions or reagents will induce the reprogramming process in the cow, we decided to take a different approach. We focused on trying to understand how nuclear reprogramming works in the bovine. This would allow us to rationally target the specific requirements of potential bovine pluripotent cells. It is known that the fusion (“merging”) of an adult cell with a stem cell, causes the adult cell to change its gene expression pattern to resemble a stem cell. We therefore fused mouse ESCs with bovine fibroblasts, and observed changes in bovine gene expression pattern as early as 24 hours after fusion. The gene expression changes observed resemble those found during early reprogramming of human and mouse iPSCs, and are accompanied by silencing of fibroblast specific genes. This suggests that our cell fusion model recreates the changes that happen during reprogramming, and can therefore be used as a tool to better understand pluripotency in the cow. The cell fusion method described in this dissertation can in theory be adapted to other species; by fusing somatic cells from other species to mouse ESCs, this model can be used to find species specific relevant pluripotency genes.

Master of Agribusiness / Department of Agricultural Economics / Vincent Amanor-Boadu / As the dairy industry faces the future, consumers’ demand for better milk quality and safety is increasing. Milk quality is of major interest to both consumers and dairy farmers alike. However, scientific data on milk quality in terms of somatic cell count (SCC) in Uganda and most developing countries has been lacking. This study used SCC to compare Southwestern Uganda’s milk quality against international standards. The study also sought to assess dairy farmers’ perceptions about milk quality. Milk samples were obtained from 100 farms in Mbarara and Kiruhura districts, the major cattle corridor in Uganda. The milk’s SCC was analyzed using a DeLaval DCC. A structured questionnaire surveyed farmers on milking procedures and milk-quality perception. Descriptive statistics and qualitative analysis was used to characterize and compare milk quality against the international benchmark. The study found that the 100 farms had an average SCC of 507,000 cells/ml. About 34% of farms in the study had SCC under 200,000 cells/ml, an indication of high-quality milk. Excluding 7% of the farms with SCC over 1,000,000 cells/ml, the remaining 93% had an average SCC of 276,000 cells/ml, a level comparable to international standards, well below the EU threshold of 400,000. The study also revealed that 98% of farmers considered milk quality as important or very important both to them and to the milk buyers. However, all farmers reported that they currently do not receive a milk-quality premium and are not penalized for poor quality. Seventy-nine percent of farmers reported the cooperative they belong to as their main source of information on management practices. An improved perception of milk quality both domestically and internationally will benefit Uganda’s dairy farmers and its dairy industry at large. Consumers must be assured that Uganda’s dairy industry, its government, industry stakeholders such as the Dairy Development Authority, the Uganda National Bureau of Standards, and the private sector place the utmost importance on the quality and safety of milk and other dairy products. New technologies to measure for SCC and strict food safety regulations will help improve the country’s milk-quality image, allowing Uganda’s dairy industry to tap into major milk export markets. Most developed countries have seen increased raw-milk quality or reduced SCC as a result of strong regulatory pressure.