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Bovine mastitis caused by Streptococcus uberis is a common problem in pasture-based dairying systems. This study examines both the ecology of S. uberis and infection of the bovine mammary gland on a New Zealand dairy farm. Initially, the REP-PCR strain typing method was developed and the potential of MALDI-TOF mass spectrometry evaluated as a strain typing method. While strain-specific mass spectra were obtained with MALDI-TOF mass spectrometry, the irreproducibility of spectra was its major downfall. With further work, this rapid method could be very useful for strain typing S. uberis on a large scale. Using optimised REP-PCR and anchored typing methods, multiple S. uberis strains were isolated and strain typed from the dairy environment, including farm races and paddocks, faeces, teat skin, the cow body and from intramammary infections. High strain diversity was observed in all sampled locations; however, some strains were found at more than one site, suggesting transmission may occur between the environment and cows. The most likely means of S. uberis distribution throughout the dairy farm was via excretion with faeces and, although not all cow faeces contained this pathogen, the gastrointestinal tract of some cows appeared to be colonised by specific strains, resulting in persistent shedding of this bacteria in the faeces. Infection of the mammary gland is likely to occur through contamination of the teat skin with highly diverse environmental strains of S. uberis. However, only one or two strains are generally found in milk from mastitis cases, suggesting that, although infection may arise from a random or opportunistic event, a strain selection process may take place. Intramammary challenge with multiple strains of S. uberis revealed that selection of a single infective strain can occur within the mammary gland. The predominance of one strain over others may be related to production of virulence factors allowing enhanced ability to establish in the gland and evade the immune response, or due to direct competition between strains through the production of antimicrobial factors such as bacteriocins. In addition to strain-specific factors, the individual cow and quarter response may play an important role in the development of infection and selection of the infective strain. Using results from this study, a model of S. uberis strain transmission has been proposed, which includes potential mechanisms of infection and persistence of S. uberis within the mammary gland.

Bacterial and fungal culturing was conducted on samples taken from the vaginal fornix of 106 cows, of which 42 had vaginitis and 64 had normal vaginas. The diagnosis of vaginitis and non-vaginitis samples was determined by histologic examination. Aerobic, anaerobic, and microaerophilic cultures were done. In addition, cultures were performed for Campylobacter sp., Ureaplasma sp., Mycoplasma sp., Tritrichomonas foetus, and fungi. All 106 samples contained mixed aerobic bacterial cultures. The more frequent aerobic isolates included Acinetobacter lwoffii, Arcanobacterium pyogenes, Escherichia coli, Corynebacterium spp., and Streptococcus spp. These organisms were isolated from both groups of cows, but more frequently from the vaginitis group. Anaerobic isolates included Peptostreptococcus spp., Prevotella spp., and Fusobacterium spp. The fungal isolates included Aspergillus sp., Mucor sp., and Penicillium sp.

In vitro produced (IVP) embryos not hatching from the zona pellucida (ZP) after transfer is one possible contributing factor of a lower pregnancy rate when compared to in vivo embryos. This study evaluates using a microscope objective mounted laser to cut the ZP prior to transfer into the recipient to assist hatching. The preliminary data evaluated the effect of laser treatment on IVP embryos and subsequent blastomere survival. In six replicates, bovine oocytes were in vitro produced according to the standard laboratory procedures of TransOva Genetics, Sioux Center, IA. On days 5, 6, and 7 of in vitro culture, embryos were randomly divided into 3 groups: no treatment (Control; n=63), sham ZP cut (Sham; n=68), or ZP cut (Cut; n=70). Control embryos were immediately returned to the incubator. Sham embryos were exposed to all conditions as Cut except laser assisted hatching. The XyClone® system was used to treat the Cut group using pulse strength of 90% and pulse length of 600 μsec. Embryos were returned to culture until day 8 when embryonic development and the percentage of live cells were determined and analyzed with Chi square. The number of developing embryos and the percentage of live cells per embryo showed no significant difference. Mean live cells ranged from 89-96% regardless of day of treatment. The laser assisted hatching effect on IVP embryo viability was evaluated by randomly dividing commercially produced embryos obtained from TransOva Genetics into two groups on day of transfer, Control or Cut. The ZP of treated embryos were cut with the laser using 80% pulse strength and pulse length of 500 μsec on day 7, immediately prior to transfer into estrous synchronized recipients. Ultrasonagraphy determined pregnancy rates. Thirty day pregnancy rates were 49.2% and 54.1% for Control (n= 189) and Cut (n=148) embryos, respectively, and were not statistically different (p > 0.05). However, 60 day Control pregnancy rate was 45.7% (n= 166) and the Cut group rate was 57.7% (n= 123) revealing a statistical difference (p < 0.05). These results demonstrate that the XyClone® system assisted hatching can improve 60 day pregnancy rates for IVP embryos by approximately 11 %.

The overall objective of this thesis was to investigate the vitrification of bovine cumulus oocyte complexes (COCs) as a tool for conservation of female genetics. Specifically, the first objective was to compare in vitro maturation rates (i.e. end point Metaphase II (MII) rate) of bovine COCs following vitrification using two different equilibration times (0 vs 10 min) in vitrification solution 1 (VS1) and two different cryodevices (cryotop vs 0.25 mL straw). The MII rate was higher in the non-vitrified control group than in vitrified groups (61 vs 16%, P<0.001). In the vitrified groups, equilibration time in VS1 [TCM-199 + 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (DMSO) + 20% calf serum (CS)] did not affect MII rate (P=0.964); however, MII rate was higher in COCs vitrified on cryotops than in straws (23 vs 9%, P=0.007).<p> The second objective of this thesis was to compare cleavage and subsequent developmental competence of bovine COCs vitrified using different vitrification solutions (fresh vs frozen) and different equilibration times in VS1 (0 vs 5 min). Immature bovine COCs were vitrified on cryotops using vitrification solutions either prepared fresh or frozen/thawed, and two equilibration times in VS1 (0 vs 5 min). Cleavage and blastocyst production rates were higher (P<0.001) in the non-vitrified control group than vitrified groups (cleavage rate 93 vs 42% and blastocyst rate 31 vs 0.4%). The cleavage rate of COCs vitrified in frozen/thawed solutions with 5 min equilibration was higher (P=0.05) than in other treatment groups. However, the blastocyst rate did not differ (P=0.993) among vitrified groups.<p> We hypothesized that the nuclear stage of bovine COCs at the time of vitrification will affect post-warming in vitro maturation (IVM), cleavage and embryo development. Firstly, a preliminary study was conducted to validate our in vitro maturation system. The COCs were at germinal vesicle (GV, 89%), germinal vesicle breakdown (GVBD, 47%), metaphase I (MI, 90%), and metaphase II (MII, 84%) stages at 0, 6, 12 and 22 h of IVM respectively. In a subsequent study, bovine COCs were vitrified after 0, 6, 12 and 22 h of IVM by loading on cryotops and plunging in liquid nitrogen. Following 1 min in warming solution (TCM-199 + 17% sucrose + 20% CS), COCs were placed in IVM medium to complete 22 h of IVM and nuclear stages were evaluated using lamin A/C-DAPI staining. The nuclear maturation (MII) rates of COCs were 23, 23, 35 and 89% (P<0.001) in the 0, 6, 12 and 22 h IVM groups, respectively. In the final nuclear stage experiment, cleavage and embryo development was determined after vitrification of COCs after 0, 12 and 22 h of IVM. The cleavage and blastocyst rates were higher (P<0.001) in the non-vitrified (control) group than vitrified groups (73 vs 15% and 22 vs 0.3%, respectively). The cleavage rates (14% in 0 h, 17% in 12 h and 14% in 22 h groups; P=0.825) and blastocyst rates (0% in 0 and 22 h and 1% in 12 h groups) did not differ. Results from this study indicated that a greater proportion of COCs progressed to MII if vitrification occurred at MI rather than at GV and GVBD stages. However, the nuclear status of vitrified COCs appeared to have no effect on fertilization or subsequent embryo development.<p> A final set of experiments were designed to determine if the exposure of bovine COCs to cryoprotectant solutions and different warming time intervals affects their ability to become fertilized, cleave and develop into blastocysts. In both experiments, the cleavage and blastocyst rates in the vitrified group were lower (P<0.001) than in the non-vitrified control groups VS1 group, and in the VS1 + vitrification solution 2 (VS2; TCM-199 + 15% EG + 15% DMSO + 20% CS) group. However, the cleavage and blastocyst rates did not differ (P>0.05) in control, VS1 and VS1+VS2 groups. In the second experiment, there was no difference in cleavage rates between 1 and 5 min intervals in the warming solution. In conclusion, cryotop can be used as a preferred cryodevice for vitrification of bovine COCs. Vitrification solutions (VS1 and VS2) and the warming solution can be prepared and stored in the freezer (-20 °C) for convenience. The pre-equilibration of bovine COCs in VS1 had no effect on the nuclear maturation of vitrified/warmed COCs, but 5 min pre-equilibration in VS1 improved cleavage rates. The nuclear stage of COCs at the time of vitrification and cryoprotectant exposure appeared to have no effect on subsequent cleavage. Furthermore, there was no difference in cleavage and blastocyst rates following vitrification and 1 or 5 min warming time intervals. When compared to non-vitrified controls, the vitrification of immature bovine COCs resulted in reduced nuclear maturation, cleavage and embryo development rates. Further studies are needed to elucidate the causes of the poor embryo development in vitrified-warmed bovine COCs at genomic, proteomics and metabolomics levels.

The overall objective of this thesis was to investigate the vitrification of bovine cumulus oocyte complexes (COCs) as a tool for conservation of female genetics. Specifically, the first objective was to compare in vitro maturation rates (i.e. end point Metaphase II (MII) rate) of bovine COCs following vitrification using two different equilibration times (0 vs 10 min) in vitrification solution 1 (VS1) and two different cryodevices (cryotop vs 0.25 mL straw). The MII rate was higher in the non-vitrified control group than in vitrified groups (61 vs 16%, P<0.001). In the vitrified groups, equilibration time in VS1 [TCM-199 + 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (DMSO) + 20% calf serum (CS)] did not affect MII rate (P=0.964); however, MII rate was higher in COCs vitrified on cryotops than in straws (23 vs 9%, P=0.007).<p> The second objective of this thesis was to compare cleavage and subsequent developmental competence of bovine COCs vitrified using different vitrification solutions (fresh vs frozen) and different equilibration times in VS1 (0 vs 5 min). Immature bovine COCs were vitrified on cryotops using vitrification solutions either prepared fresh or frozen/thawed, and two equilibration times in VS1 (0 vs 5 min). Cleavage and blastocyst production rates were higher (P<0.001) in the non-vitrified control group than vitrified groups (cleavage rate 93 vs 42% and blastocyst rate 31 vs 0.4%). The cleavage rate of COCs vitrified in frozen/thawed solutions with 5 min equilibration was higher (P=0.05) than in other treatment groups. However, the blastocyst rate did not differ (P=0.993) among vitrified groups.<p> We hypothesized that the nuclear stage of bovine COCs at the time of vitrification will affect post-warming in vitro maturation (IVM), cleavage and embryo development. Firstly, a preliminary study was conducted to validate our in vitro maturation system. The COCs were at germinal vesicle (GV, 89%), germinal vesicle breakdown (GVBD, 47%), metaphase I (MI, 90%), and metaphase II (MII, 84%) stages at 0, 6, 12 and 22 h of IVM respectively. In a subsequent study, bovine COCs were vitrified after 0, 6, 12 and 22 h of IVM by loading on cryotops and plunging in liquid nitrogen. Following 1 min in warming solution (TCM-199 + 17% sucrose + 20% CS), COCs were placed in IVM medium to complete 22 h of IVM and nuclear stages were evaluated using lamin A/C-DAPI staining. The nuclear maturation (MII) rates of COCs were 23, 23, 35 and 89% (P<0.001) in the 0, 6, 12 and 22 h IVM groups, respectively. In the final nuclear stage experiment, cleavage and embryo development was determined after vitrification of COCs after 0, 12 and 22 h of IVM. The cleavage and blastocyst rates were higher (P<0.001) in the non-vitrified (control) group than vitrified groups (73 vs 15% and 22 vs 0.3%, respectively). The cleavage rates (14% in 0 h, 17% in 12 h and 14% in 22 h groups; P=0.825) and blastocyst rates (0% in 0 and 22 h and 1% in 12 h groups) did not differ. Results from this study indicated that a greater proportion of COCs progressed to MII if vitrification occurred at MI rather than at GV and GVBD stages. However, the nuclear status of vitrified COCs appeared to have no effect on fertilization or subsequent embryo development.<p> A final set of experiments were designed to determine if the exposure of bovine COCs to cryoprotectant solutions and different warming time intervals affects their ability to become fertilized, cleave and develop into blastocysts. In both experiments, the cleavage and blastocyst rates in the vitrified group were lower (P<0.001) than in the non-vitrified control groups VS1 group, and in the VS1 + vitrification solution 2 (VS2; TCM-199 + 15% EG + 15% DMSO + 20% CS) group. However, the cleavage and blastocyst rates did not differ (P>0.05) in control, VS1 and VS1+VS2 groups. In the second experiment, there was no difference in cleavage rates between 1 and 5 min intervals in the warming solution. In conclusion, cryotop can be used as a preferred cryodevice for vitrification of bovine COCs. Vitrification solutions (VS1 and VS2) and the warming solution can be prepared and stored in the freezer (-20 °C) for convenience. The pre-equilibration of bovine COCs in VS1 had no effect on the nuclear maturation of vitrified/warmed COCs, but 5 min pre-equilibration in VS1 improved cleavage rates. The nuclear stage of COCs at the time of vitrification and cryoprotectant exposure appeared to have no effect on subsequent cleavage. Furthermore, there was no difference in cleavage and blastocyst rates following vitrification and 1 or 5 min warming time intervals. When compared to non-vitrified controls, the vitrification of immature bovine COCs resulted in reduced nuclear maturation, cleavage and embryo development rates. Further studies are needed to elucidate the causes of the poor embryo development in vitrified-warmed bovine COCs at genomic, proteomics and metabolomics levels.

Waves of bovine genomic data have been produced as a result of the bovine genome sequencing projects. In addition to the massive amounts of genomic sequence, significant annotation including single nucleotide polymorphisms, sequence tagged sites and haplotype blocks have been produced by the Bovine HapMap Project. Furthermore, many agriculturally significant traits in cattle such as milk yield and carcass weight are measured on a quantitative scale and have been genetically mapped as quantitative trait loci (QTL). QTL data can be used to generate another form of bovine annotation linking phenotype to genotype. However, it is impossible for humans to be able to analyze genomic scale data without computer based tools. Bioinformatic tools have been shown to greatly increase productivity and improve efficiency when dealing with large data sets. My dissertation presents an integrated, extensible database that houses SNPs, STSs, haplotypes, and QTL. The database is presented to researchers through a restructured, object oriented Bovine QTL Viewer that displays multiple levels of bovine annotation synergistically. Evaluation of use of the viewer was performed using a survey based approach and measured quantitatively. In addition, the QTL data from the database was used to analyze the frequency of gene ontology (GO) annotations within QTL regions. QTL regions were divided into 8 trait based groups. GO terms were counted within each category of QTL and in non- QTL regions of the genome. Top level GO term frequencies were generated from the counts and these frequencies were compared between QTL and non-QTL portions of the genome. Furthermore, specific sets of GO terms believed to be related to QTL categories were also used to determine if QTL regions were enriched for genes annotated with such GO terms. As a result, we determined that gene density varied significantly across QTL regions and that many QTL categories showed GO term frequency differences that could be related to the trait’s biology. Furthermore, our selected GO term sets were shown to be significantly enriched in some QTL categories.

The ability to create transgenic livestock is a tremendous benefit in scientific research for many disciplines including functional genomics, pharmaceutical synthesis and development of enhanced production animals. Transgenes can either be stably or transiently expressed to alter gene function and obtain a specifically engineered phenotype. To create a transgenic bovine embryo, genetically altered somatic cells must be used in somatic cell nucleus transfer, or early 1-cell embryos (zygotes) must be microinjected with plasmid DNA or small interfering RNA (siRNA). Given the cost and skill associated with both methods, a preliminary investigation exploring alternative delivery techniques of siRNA (transient expression) into bovine zygotes with a nonhomologous Cy3 labeled siRNA (Cy3-siRNA) was first performed. It was discovered that zygotes injected with more than 50 Bmol L-1 of Cy3-siRNA fail to form a blastocoel and that, although bovine zygotes are not susceptible to chemical transfection, the trophectoderm cells of the blastocyst are. Based on this information, bovine E-cadherin gene expression was compared in day 9 blastocysts derived from either injected zygotes (day 1) or transfected blastocysts (day 7) with a Cy3 labeled E-cadherin specific siRNA (Cy3-siEcad) to determine 1) if gene suppression in zygotes injected with 25 Bmol L-1 Cy3-siEcad continues during embryo development up to hatching, and 2) if blastocysts transfected at a ratio of 9:6 with GeneJammer® truly experience gene knock down after siRNA transfection capable of maintaining suppression to day 9. Quantitative PCR indicated blastocysts transfected with Cy3-siEcad had a significant 15.3% decrease (P < 0.05) in E-cadherin mRNA at day 9 compared to the injected zygotes. Protein fluorescence analysis from immunocytochemistry of whole mounted day 9 blastocysts revealed injected zygotes accumulated significantly less E-cadherin protein (67.7%) than the transfected blastocysts (P < 0.05). From these data, it can be concluded that although siRNA injection may be capable of knocking down gene expression for the first 7 days of embryonic development, it does not persist to the hatching stage; however, blastocysts transfected at day 7 do express altered gene expression in the trophectoderm which can continue through embryonic hatching events.

In vitro produced (IVP) embryos not hatching from the zona pellucida (ZP) after transfer is one possible contributing factor of a lower pregnancy rate when compared to in vivo embryos. This study evaluates using a microscope objective mounted laser to cut the ZP prior to transfer into the recipient to assist hatching. The preliminary data evaluated the effect of laser treatment on IVP embryos and subsequent blastomere survival. In six replicates, bovine oocytes were in vitro produced according to the standard laboratory procedures of TransOva Genetics, Sioux Center, IA. On days 5, 6, and 7 of in vitro culture, embryos were randomly divided into 3 groups: no treatment (Control; n=63), sham ZP cut (Sham; n=68), or ZP cut (Cut; n=70). Control embryos were immediately returned to the incubator. Sham embryos were exposed to all conditions as Cut except laser assisted hatching. The XyClone® system was used to treat the Cut group using pulse strength of 90% and pulse length of 600 μsec. Embryos were returned to culture until day 8 when embryonic development and the percentage of live cells were determined and analyzed with Chi square. The number of developing embryos and the percentage of live cells per embryo showed no significant difference. Mean live cells ranged from 89-96% regardless of day of treatment. The laser assisted hatching effect on IVP embryo viability was evaluated by randomly dividing commercially produced embryos obtained from TransOva Genetics into two groups on day of transfer, Control or Cut. The ZP of treated embryos were cut with the laser using 80% pulse strength and pulse length of 500 μsec on day 7, immediately prior to transfer into estrous synchronized recipients. Ultrasonagraphy determined pregnancy rates. Thirty day pregnancy rates were 49.2% and 54.1% for Control (n= 189) and Cut (n=148) embryos, respectively, and were not statistically different (p > 0.05). However, 60 day Control pregnancy rate was 45.7% (n= 166) and the Cut group rate was 57.7% (n= 123) revealing a statistical difference (p < 0.05). These results demonstrate that the XyClone® system assisted hatching can improve 60 day pregnancy rates for IVP embryos by approximately 11 %.

Sex pre-selection in livestock offspring to produce dairy cows and specific male and females lines for heterosis in swine are important economic goals in animal husbandry. The long-term goal of the study is to separate porcine and bovine chromosome X- or Y-bearing sperms using sex-specific antibodies. H-Y antigen encoded by jumonji, AT-rich interactive domain 1d (Rbp2-like) (Jarid1d) is defined as a male histocompatibility antigen that causes rejection of male skin grafts by female recipients of the same inbred strain of rodents. Porcine and bovine JARID1D genes are estimated between 3.5 kb and 4.5 kb, however there was only 457 bp and 99 bp respectively found in the database. In this study, the porcine and bovine sex-specific genes, JARID1D were cloned using in silico cloning, PCR and cDNA library screening. We used human JARID1D gene as a probe or template to search the ESTs of porcine and bovine in NCBI and TIGR databases and combined the results from two databases and assembled the ESTs using CAP3 program. We also constructed porcine and bovine testis cDNA libraries, and performed porcine testis cDNA libraries screening. To study this model in the mouse, Jarid1d H-Y epitope TENSGKDI was synthesized and used to immune the rabbits. Dot blotting analysis revealed the specificity of the immuned antibodies to mouse Jarid1d H-Y epitope TENSGKDI, but western blotting analysis did not confirm the results. Immunofluorescence analysis of mouse sperms reacted with anti-mouse Jarid1d H-Y epitope TENSGKDI-specific antibody didn¡¦t show a 1:1 ratio of stained sperms to unstained sperms. The specificity of this antibody needed to be improved. The data shows partial JARID1D gene of pig was cloned, and the anti-mouse Jarid1d H-Y epitope TENSGKDI-specific antibodies were prepared, however the specificity of anti-mouse Jarid1d H-Y epitope TENSGKDI-specific antibodies to surface protein expression on mouse sperms needs more confirmations.