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The development of somatic nuclear transfer procedures and the factors that are likely to affect the success of nuclear transplantation were discussed in the first chapter. In chapter 2, the effects of cell cycle stage and age of the cell donor animal on in vitro development of bovine nuclear transfer embryos were investigated. Cultures of primary bovine fibroblasts were established from animals of various ages and the in vitro life span of these cell lines were analyzed. The cell cycle characteristics of both fetal and adult cells were analyzed as a population of cells in culture. To study the individual cells from a population, a “shake off” method was developed to isolate G1 cells and evaluate progression through cell cycle. Irrespective of the age of the cell donor, the mean cell cycle length in isolated cells was shorter than what was observed for cells cultured as a population. In vitro development was analyzed after fusing confluent and isolated G1 cells to enucleated metaphase II oocytes. These results indicate that there were no differences in either the number of cells in blastocysts or the percentage of blastocysts between the embryos reconstructed with G1 cells and confluent cells. Because previous studies do not clearly demonstrate the cell cycle stage of the donor cell that resulted in development to term, Chapter 2 evaluates the methods of producing populations of G0 and G1 cells and the cell cycle characteristics of G0 and G1 cells. Furthermore, in vitro and in vivo development of embryos derived from high confluent, G0 cells and “shake off” G1 cells were evaluated. These results indicate that the isolated G1 cells supported development to term better than confluent cells. Finally, in Chapter 3, pregnancy establishment, pregnancy loss and health of newborn calves were evaluated for nuclear transfer embryos derived from cells of different genotypes from dairy and beef breeds. In addition, the effect of overnight shipping of cloned embryos and the synchrony between the age of the embryo and the estrus cycle of the recipient female on development were assessed. The results indicate that genotype has an effect on development of nuclear transfer embryos.

WC1 molecules are an extensively diversified family of integral type 1 scavenger receptor cysteine rich (SRCR) membrane glycoproteins expressed on the surface of arteriodactyls such as sheep, cattle, and swine. The focus of this dissertation is on establishing the relevance of different forms of WC1 expressed on the surface of bovine γδT cells. The results demonstrate that identification of WC1 variant expression on these lymphocytes can be used to determine cellular responses to characterized methods of activation in vitro. In this way, discrete forms of WC1 are shown to be critical antigenic determinants for functionally distinct populations as demonstrated via CD25 expression, proliferation, and production of the inflammatory cytokine IFN-γ. At present, WC1 is the only unique determinant found on γδT cells other than the TCR that can be used to distinguish such populations. Genomic analysis carried out by others suggested the potential for more than 50 forms of WC1. This work is the first to show numerous biochemically distinct forms of plasma membrane-bound WC1 using 2D transfer methods. Currently, the bovine genome is sequenced but unassembled. Once complete, the procedures employed here can be used in conjunction with the genomic data and mass spectrometry to identify amino acid sequences of immunoprecipitated WC1 separated via 2D methods.

Equine laminitis is a crippling disease affecting up to 1% of horses in USA. Treatments are typically unsuccessful, due to a lack of effective therapeutics, which reflects our lack of understanding of the pathophysiology of the disease. There are three prominent theories regarding the initial events leading to lamellar pathology: ischemia-reperfusion (I/R) injury, alterations in laminar metabolism and systemic inflammation. It is considered that matrix metalloproteinases (MMP) may contribute to tissue damage, irrespective of events governing the onset of laminitis. Two experimental models of laminitis were utilized, the black walnut extract (BWE) model, and the carbohydrate overload model (CHO). These model systems were used to examine the contributions of I/R, inflammation and MMPs to the development of laminitis. In the BWE model, we found no biochemical indicators of I/R injury, namely no conversion of xanthine dehydrogenase to xanthine oxidase. As early as 1.5 hours post-BWE administration, there was accumulation of proMMP-9, infiltration of neutrophils into the laminae and induction of genes encoding proinflammatory cytokines (e.g IL-1β, IL-8 and IL-6). Concentrations of proMMP-9 increased over time through to Obel grade 1 lameness (OG1), while proMMP-2 and MMP-2 did not change relative to controls. In CHO-induced laminitis, levels of proMMP-2 and MMP-2 remained similar to controls through OG1, but there was a significant accumulation of MMP-2 at later stages of the disease. ProMMP-9 was detectable as early as OG1, and was highly elevated at Obel grade 3 lameness (OG3). MMP-9 and MMP-2 were elevated to varying degrees in naturally acquired laminitis depending on the phase of the disease examined, being highest in animals with aggravated chronic laminitis. Lamellar ProMMP-9 correlated positively with myeloid cells, the majority of which are neutrophils during the stages of laminitis investigated. Despite the increases in gelatinase levels, all of which were shown by SDS-PAGE and zymography, there was no overall increase in native gelatinase activity relative to controls, suggesting association of MMP with tissue inhibitors of MMP (TIMPs). These results suggest that inflammation may play a role in the development of pathology in laminitis but raise questions regarding the contributions of MMP-9 and MMp-2 to tissue damage.

The Smyth line chicken serves as a biomedical model for the human disease vitiligo. In the chicken, this condition results in white feathers and a disruption of the retinal pigmented epithelium of the eye, which can lead to visual impairment. The expressivity of this amelanosis is highly variable and does not follow single gene inheritance patterns. This dissertation addresses genetic and endocrinological factors which affect either the melanocyte or the immune system and their effect on the incidence, onset time and severity of the amelanosis. Techniques utilized in this dissertation to investigate amelanotic inheritance included genetic outcrosses, backcrosses and selection criteria. The administration routes of the treatments on this work included IM and IV injections, implantation of silastic tubing and mini-osmotic pumps and incorporation into the feed. Birds were monitored for immune system response, serum glucose levels and rate of gain as well as the development of both feather amelanosis and visual impairment. Treatments were performed on embryos, chicks, juveniles and adult Smyth and control line individuals. The results indicate that the variability in the Smyth line amelanosis can be explained by many physiological and genetic factors. An increase in pigmentation intensity will increase the incidence and severity of amelanosis in the offspring. Corticosterone and testosterone administration resulted in a significant depression in the incidence and severity of amelanosis. One haplotype (EA-B/101) at the major histocompatibility locus of the chicken, exhibits a significantly higher incidence and severity of amelanosis and visual impairment. Several of the flocks in these trials exhibited a significant sex effect (females $>$ males) in amelanotic incidence. It is concluded that the amelanotic variability in the Smyth line is due to individual physiological differences between birds due to sex, environment, health and genetics. It is apparent that any condition which yields a generalized suppression of the immune response will result in a decreased incidence of amelanosis.

In Chapter 1 we characterized the frequency, amplitude and duration of (Ca$\sp{2+}\rbrack\sb{\rm i}$ rises in fertilized rabbit eggs loaded with fura-2 dextran. Their amplitude decreased and duration increased as fertilization progressed. Injection of 1,4,5 inositol trisphosphate (InsP3; IICR) or addition of thimerosal elicited (Ca$\sp{2+}\rbrack\sb{\rm i}$ rises which were blocked by heparin, an InsP3 receptor antagonist. Ryanodine did not evoke Ca$\sp{2+}$ release. These results indicate that IICR is likely stimulated during fertilization. Fertilization (Ca$\sp{2+}\rbrack\sb{\rm i}$ changes were examined in in vitro matured bovine eggs (Chapter 2). Fertilized eggs exhibited different intervals between (Ca$\sp{2+}\rbrack\sb{\rm i}$ rises which ranged from 15 min to more than 60 min. Unfertilized eggs were responsive to InsP3 injection and thimerosal exposure, although the frequency of the (Ca$\sp{2+}\rbrack\sb{\rm i}$ responses was shorter than the periodicity observed during fertilization. The mechanisms that generate (Ca$\sp{2+}\rbrack\sb{\rm i}$ oscillations were examined in fertilized rabbit eggs (Chapter 3). Heparin blocked or delayed (Ca$\sp{2+}\rbrack\sb{\rm i}$ oscillations. Oscillating eggs exhibited Ca$\sp{2+}$ release in response to CaCl2 injection. In unfertilized eggs, injection of GTP (S) induced (Ca$\sp{2+}\rbrack\sb{\rm i}$ oscillations and enhanced the response to CaCl2 injection. Injection of InsP3 or CaCl2 elicited full Ca$\sp{2+}$ responses that reset the periodicity of ensuing oscillations. Thus, IICR participates in the generation of (Ca$\sp{2+}\rbrack\sb{\rm i}$ oscillations and the unloading of the stores does not explain the interval between (Ca$\sp{2+}\rbrack\sb{\rm i}$ rises. The signaling pathways possibly stimulated by the sperm during fertilization were investigated in unfertilized bovine eggs (Chapter 4). Injection of GTP (S) or InsP3 evoked (Ca$\sp{2+}\rbrack\sb{\rm i}$ oscillations. Preinjection of heparin blocked sperm-mediated egg activation. Thimerosal elicited (Ca$\sp{2+}\rbrack\sb{\rm i}$ oscillations which were not inhibited by heparin or ryanodine. The data suggest that bovine eggs possess a GTP-linked phosphoinositide pathway which appears to be stimulated by the sperm during fertilization. In Chapter 5 the amplitude and duration of (Ca$\sp{2+}\rbrack\sb{\rm i}$ rises elicited by electrical pulses of different strengths and duration administered in variable extracellular Ca$\sp{2+}$ concentrations was reported. As these parameters increased, so did the peak (Ca$\sp{2+}\rbrack\sb{\rm i}$ elicited by the pulse. Young and aged eggs exhibited similar (Ca$\sp{2+}\rbrack\sb{\rm i}$ rises when stimulated with identical pulses. However, their activation rates were different. Thus, aged eggs are more sensitive to a given (Ca$\sp{2+}\rbrack\sb{\rm i}$ rise.