Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / This thesis encompasses work that aimed to further understand genomic reprogramming, an event crucial in obtaining development in cloned embryos produced by somatic cell nuclear transfer (SCNT). An increasing number of different mammalian species have been cloned using nuclear transfer technology since Dolly the cloned sheep was first successfully produced. However, the biological mechanisms involved in the process of nuclear reprogramming are yet to be fully described. At the centre of this study was an intergeneric SCNT model, which was implemented to determine whether reprogramming factors are conserved across genera. The interaction between donor nucleus and recipient ooplasm was characterised with regard to developmental potential, timing of genome activation, nucleolus formation, and expression of significant proteins. In initial studies, fusion parameters of the intergeneric SCNT procedure were optimised for the ovine cytoplast and porcine donor granulosa cell. Cell fusion and lysis percentages were determined over a range of electrical pulse voltage, duration and repetition. The optimal electrofusion settings were a single DC pulse of 1.5 kV/cm for 20 usec following a 2 sec 400 kHz alignment pulse. In addition, it was demonstrated that ovine oocytes were sensitive to electric stimulation to the extreme that oocyte activation would occur no matter how low the voltage. The practical significance was that it would not be possible to implement a fusion before activation protocol. The ability of the ooplasm of one species to replicate chromosomes and support early embryo cleavage was determined in a preliminary experiment where intergeneric embryos were produced by SCNT using bovine and ovine foetal fibroblasts, and ovine ooplasm. After their construction, the embryos were allowed to develop for 7 days in vitro and the developmental stage determined by Hoechst staining and nuclei counting. In addition, chromosome spreads of the ovine and bovine somatic foetal fibroblast cell lines used in SCNT, as well as the intra- and intergeneric SCNT embryos were prepared to determine whether the ovine ooplasm was replicating the chromosomes according to the karyotype of the donor nucleus. The somatic cells were karyotyped with 54 and 60 chromosomes counted for ovine and bovine cells respectively. Bovine-ovine embryos were characterised as having a bovine karyotype as distinct from an ovine karyotype, due to the presence of only two metacentric chromosomes as compared with six that are found in the latter. These preliminary results indicated that bovine nuclei obtained from foetal fibroblast cells could initiate early pre-implantation embryo development with the support of ovine oocyte cytoplasm. The development of a proportion (33%) of ovine-ovine intrageneric SCNT embryos beyond the 16-cell stage indicated that an extensive characterisation of an intergeneric model could be performed satisfactorily. It was hypothesised that the ovine ooplasm would possess the ability to direct in vitro preimplantation embryo development after nuclear transfer using donor nuclei from a different genus, as has been demonstrated in studies using bovine and rabbit ooplasm. In this study, intergeneric SCNT embryos were constructed by the separate fusion of porcine and bovine cells with ovine cytoplasts (bovine-ovine and porcine-ovine respectively), cultured in vitro and the developmental characteristics compared with ovine-ovine SeNT embryos as well as ovine in vitro produced (IVP) embryos. These four groups of embryos were sampled to determine embryo cell numbers at 24, 36, 48, 72, 96, 120 and 168 h post-activation to compare development over time. Despite cleaving normally and undergoing the first three cleavage divisions at a rate comparable with ovine-ovine SCNT embryos, a major block in development occurred in the intergeneric embryos at the 8-16 cell stage. Consequently, no blastocyst formation was obtained as observed for the IVP and ovine-ovine SCNT controls. These results indicate that unlike the rabbit and bovine ooplasm, the ovine ooplasm is not suitable for intergeneric reprogramming of somatic nuclei from another genus, at least of porcine or bovine origin. To determine the effect of a less differentiated donor nucleus on intergeneric developmental potential, embryonic cell nuclear transfer (ECNT) was conducted in a separate experiment by fusing pluripotent bovine and ovine donor cells (obtained from day-4 preimplantation embryos) to ovine cytoplasts. After 7 days of culture, the cell number of embryos was determined by Hoechst staining and fluorescent observation. Despite observing a single bovine-ovine blastocyst (4.8%), the developmental block remained at the 8-16 cell stage of development. This outcome indicates that a less differentiated nucleus does not increase intergeneric developmental capability. It is well documented that the ooplasm supplies a large amount of mRNA and protein to the newly formed embryo, crucial for normal development leading up to the major activation of the embryonic genome. However, the interaction between the ooplasm as compared with the donor nucleus in SCNT embryos during this developmental period is poorly understood. This intergeneric SCNT model provided an opportunity to determine the role of the ooplasm on nucleolus formation, which is a marker for genome activation. Ultrastructural evidence was obtained that indicates the ovine ooplasm directs the initial assembly of the nucleolus independent of the species of the nuclear donor. Intergeneric porcine-ovine SCNT and intrageneric ovine-ovine SCNT embryos were constructed and the nucleolus ultrastructure and nucleolus associated rRNA synthesis examined in 1-,2-,4-, early 8-, late 8-and 16-cell embryos using transmission electron microscopy (TEM) and light microscopical autoradiography. Intergeneric porcine-ovine SCNT embryos exhibited nucleolar precursor bodies (NPBs) of an ovine (ruminant) ultrastructure, but no active rRNA producing fibrillogranular nucleoli at any of the stages. Unusually, cytoplasmic organelles were located inside the nucleus of two porcineovine SCNT embryos. The ovine-ovine SCNT embryos, on the other hand, revealed fibrillogranular nucleoli in 16-cell embryos. In parallel, autoradiographic labelling over the nucleoplasm and, in particular, the nulcleoli was detected. Bovine-ovine SCNT embryos at the 8-cell stage were examined for nucleolar morphology and exhibited ruminant-type NPBs as well as structures that appeared to comprise of broken down fibrillar material, perhaps formerly of nucleolar origin from the donor cell. These observations indicate that factors within the ovine ooplasm are playing a role in the initial assembly of the embryonic nucleolus in intrageneric SCNT embryos. To further characterise nucleolus formation, immunocytochemical localisation by confocal microscopy of nucleolin, fibrillarin and RNA polymerase, three key proteins involved in processing rRNA transcripts, was performed on early 8-, late 8- and 16-cell embryos for ovineovine and porcine-ovine SCNT embryos. Nucleolin was localised throughout the nucleoplasm for all developmental stages examined in porcine-ovine and ovine-ovine SCNT embryos and, in particular, intensity around the presumptive nucleolar compartments in the later developmental stages. Fibrillarin and RNA polymerase I, on the other hand, were not detected in any ovineovine or porcine-ovine SCNT embryos or ovine IVP controls, although both proteins were detected in control bovine IVP blastocysts. This result indicates that the antifibrillarin and anti-RNA polymerase I were not compatible with the ovine form of these respective proteins. As nucleolin is not present in porcine in vivo embryos before the major activation of the embryonic genome, its presence in porcine-ovine SCNT embryo nucleus indicates that nucleolin is derived from the abundant protein and mRNA stored in the ovine ooplasm. The intergeneric SCNT model established in this thesis demonstrates that the ovine ooplasm lacks the ability to support embryonic development beyond the 16-cell stage. The TEM and autoradiographical studies, in combination with the protein immunocytochemistry study, confirmed that these embryos are unable to undergo the major activation of the embryonic genome, and that the ooplasm influences the initial nucleolar assembly in these embryos. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1167553 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture and Wine, 2005

Mastitis, the inflammation of the mammary gland caused by both gram-positive and gram-negative bacteria, causes the global dairy industry over $20 billion annually in losses due to changes in milk yield and quality. The goals of this research were to 1) identify genetic variants, genes, and biological pathways that are associated with mastitis, and 2) identify genetic variants, genes, and biological pathways that are associated with somatic cell count. In Chapter 3, we describe genome-wide association studies (GWAS) on 1,858 cows from two lactations with both medium (n=87,493) and high density (n=624,300) single nucleotide polymorphisms (SNP) using a single SNP mixed linear model. A total of 289 SNPs across all models reached genome-wide significance at 5% false discovery rate (FDR) and were mapped to 100 different nearby genes which have been identified in previous studies and are quantitative trait loci for somatic cell count. Significant SNPs and genes were mapped to the bovine genome to reveal corresponding genes to allow gene enrichment analysis to identify significant biological pathways associated with mastitis. In Chapter 4, we describe genetic associations affecting lactation average somatic cell count using 624,300 single nucleotide polymorphisms (SNP) on performed on 364 Holstein and Jersey cows in their first lactation. A total of 205 SNPs reached genome-wide significance at 5% FDR and were mapped to 68 unique genes.

Somatic cell nuclear transfer (SCNT) is a powerful tool for production of transgenic animals for various biomedical and agricultural applications. For instance, our group is using SCNT to produce transgenic goats to study the role of cardiac fibrosis in initiation and progression of atrial fibrillation. There is a possibility of cell transfer from a transgenic fetus to its non-transgenic surrogate mother, known as fetal microchimerism; from a transgenic mother to non-transgenic fetus, maternal microchimerism and from a transgenic twin to non-transgenic twin in utero. Initially, we have assessed the presence of fetal microchimerism in tissue samples from non-transgenic surrogates that delivered transgenic SCNT generated offspring. Then, the SCNT derived transgenic goats were naturally bred and non-transgenic offspring were used for the assessment of maternal microchimerism. Additionally, fetal-fetal microchimerism was evaluated using the tissue samples from non-transgenic twins of transgenic offspring. We investigated DNA from kidney, liver, lung, lymph node and spleen for the presence of neomycin resistant gene (Neo), which all transgenic SCNT generated females and their transgenic offspring tested positive for. We found no detectable maternal or fetal-fetal microchimerism, but fetal microchimerism was detected in lymph node of one of the surrogate dams that carried a SCNT pregnancy. The results of the study have direct implications on the use and disposal of non-transgenic surrogates and non-transgenic offspring.

Infection data were obtained monthly from June, 1999 to September, 2000 at the University ofKentucky dairy. Quarter foremilk samples were collected for bacteriological determination andsomatic cell counts (SCC). The Livestock Stress Index (LSI) estimated heat stress and is calculatedby combination of temperature and humidity. For uninfected quarters the geometric mean SCC was29,000 cells/ml. For infected quarters the geometric mean SCC was 213,000 cells/ml. Coagulasenegativestaphylococci (CNS) infections comprised 61 percent of the total infected quarters with ageometric mean SCC of 155,000 cells/ml. Staphylococcus aureus infected quarters had a geometricmean SCC of 680,000 cells/ml. There were no significant correlations between log SCC and LSIwhen looking at the total sample period. However, evaluating October, 1999 through September,2000, significant correlations were found for LSI and log SCC of uninfected quarters (P andlt; 0.05) and`infected quarters (P andlt; 0.0001). All correlation coefficients were less than 0.12. The results suggest nomarked changes in SCC were observed in uninfected quarters during hot summer weather. Hotsummer weather may have a minor impact on SCC in infected quarters, but the effect is variable.Thus, infection status of the mammary gland, not heat stress, is the major factor determining SCC.

Objective 1 was to investigate effects of heat stress and breed on milk and component yield for Holstein and Jersey cows on the same farm. Objective 2 was to determine the effects of breed on udder health as measured by somatic cell count (SCC) during times of heat stress. Data were collected from DHIA records of 142 Jersey cows and 586 Holstein cows from the University herd at Mississippi State University. During heat stress Jersey milk yield and 4% fat corrected milk (FCM) increased (P<0.01). Holstein milk yield and FCM decreased during heat stress (P<0.01). Heat stress affected somatic cell count (SCC) although effects varied by intensity of heat stress. Breed did not have an affect on SCC. Milk fat and protein percentages declined for both breeds in heat stress conditions. Milk fat but not milk protein of Jersey cows increased as stress increased from mild to severe.

Somatic Cell Nuclear Transfer (SCNT), commonly known as cloning, is the transfer of a somatic nucleus into an enucleated oocyte to produce a clone. The chromatin structure of somatic cells permits the expression of certain genes, while silencing the rest of the genome. The cytoplasm of oocytes can reprogram a somatic nucleus by reactivating the genes necessary for embryonic development and silencing the somatic genes. However, the low efficiency of SCNT indicates that successful nuclear reprogramming is a rare event. The objectives of this study were determine the extent of transcriptional reprogramming in bovine blastocysts produced by serial rounds of chromatin transfer (from first and fourth generations), using blastocysts produced by in vitro fertilization (IVF) as controls, to identify cumulative errors in the transcriptome profile. Differentially expressed genes were studied further to determine their function in embryonic development. We identified a set of transcripts consistently misregulated in cloned blastocyst, some of which had a more marked misregulation in the embryos produced by 4 successive rounds of cloning. Among the genes significantly upregulated in both CT groups compared to IVF blastocysts were both de novo DNA methylation enzymes DNMT3A and DNMT3B. Expression patterns, structural and functional analyses were performed for DNA methyltransferases. A high structural and functional conservation was observed for DNA methyltransferases among human, mouse, and bovine species. A set of genes that participate in early embryonic development, chromatin remodeling and DNA methylation were differentially regulated in cloned embryos and had not been fully annotated at the time of the analysis. We annotated those genes and submitted them to the Bovine Genome Sequencing Consortium database. These results have important implications for the selection of models for the study of DNA methylation during early development. The present study provides a valuable data set for identifying possible cumulative errors in somatic cell chromatin transfer that could hinder nuclear reprogramming shedding light on the epigenetic role in reprogramming and cell plasticity.

No description available.

Mastitis is one of the most costly diseases to dairy producers around the world with milk yield loss being the biggest contributor to economic losses. The objective of first study of this thesis was to determine the impacts of high somatic cell counts on milk yield loss. To accomplish this, over one million cow data records were collected from Southeastern US dairy herds. The objective of the second study was to determine optimum treatment cost of clinical mastitis by combining two economic modeling approaches used in animal health economics. The last objective of this thesis was to determine how much Southeastern US dairy producers are spending to control milk quality on farm and determine if they understand how milk quality affects them economically. This was accomplished through a collaborative project within the Southeast Quality Milk Initiative.

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).

Somatic cell nuclear reprogramming (SCNR) by eggs is a way to forcibly transform the nuclei of terminally differentiated somatic cells to an embryonic state and gain totipotency (Gurdon et al., 1958). Additionally, induced pluripotency is applied to transform identities of somatic cells to induced pluripotent stem cells by overexpression of combinatorial Yamanaka factors (iPS, Takahashi et al., 2006). Although both approaches aim to derive cells with highest plasticity, the mechanisms and differences between these procedures are not yet clear. In my thesis, I used quantitative polymerase chain reaction (QPCR) and RNAseq plus 5-bromouridine 5'-triphosphate (BrUTP) pulldown to evaluate the transcriptional reprogramming by maternal factors and overexpressed transcription factors during SCNR by Xenopus oocytes, which are inactive in DNA replication and cell division. QPCR measures changes in the steady-state levels of transcripts within 2 days of nuclear transfer to Xenopus oocytes (Oocyte-NT). Three pairs of Yamanaka factor homologs were tested by QPCR and Yamanaka factor homologues regulated similar sets of pluripotency genes in mouse embryonic fibroblasts (MEFs). Pioneer factor mFoxA1 could not up-regulate most pluripotency genes and their binding targets of neurogenic genes in MEFs while pioneer factors are proposed to bind to their targets even if they may reside in inaccessible chromatin. This shows that the existence of other factors is needed at specified developmental stages. Hence, gene activation by transcription factors in the Oocyte-NT system requires not only corresponding binding on regulatory elements of linked genes but transcription cooperators to exert effective gene activation. Additionally, RNA-seq plus BrUTP pulldown measures the extent to which oocytes change the transcriptional activity of nuclei transplanted to oocytes. Through RNA-seq plus BrUTP pulldown, I compared the reprogrammed transcriptomes of embryonic and somatic cells, including mouse embryonic stem cells, mouse embryonic fibroblasts and mouse myoblasts, to demonstrate the effects of maternal factors and overexpression of transcription factors on gene activities during SCNR by oocytes. Importantly, I find that maternal factors of oocytes and the overexpression of transcription factors exert different strategies to reprogram somatic cells. Oocyte factors reprogram the donor cell nuclei to an oocyte-steady state except for the SCNR resistance genes and xklf2-HA overexpression enhances expression of reprogrammable genes and activates SCNR resistance genes.