Call number: LD2668 .T4 1957 H67 / Master of Science

Digitized by Kansas Correctional Industries

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Milk yield is dependent on both the number and secretory activity of mammary alveolar cells. The number of cells is controlled by their proliferation and death. In the first study, the effect of eukaryotic translation initiation factor 4E (eIF-4E) on the growth of a bovine mammary epithelial cell line, MAC-T, was investigated. Compared to the parental controls, overexpression of mouse wild-type (wt) eIF-4E in 11A, a subclone of MAC-T cells, increased the growth rate and saturation density (number of cells per well at confluence), whereas overexpression of a mutant eIF-4E (W56A) decreased growth rates and saturation densities. Furthermore, cyclin D1 expression among the 4E-overexpressing and parental cells was compared. Compared to the controls, the amounts of cyclin D1 mRNA and proteins were higher in the cells overexpressing wt eIF-4E but lower in the cells with mutant eIF-4E expression. Our results suggest that altered expression of eIF-4E leads to changes in cyclin D1 expression, which consequently modulate the growth properties of MAC-T 11A cells. / Because of its unknown sequence, bovine eIF-4E cDNA was then cloned in the second study. Its coding region consists of 651 nucleotides which encode 217 amino acids (AAs). Bovine eIF-4E cDNA shares 94%, 89% and 94% homology with those of human, mouse and rabbit, respectively. Differences in protein sequences between bovine and human, mouse and rabbit eIF-4E are 2, 4, and 3 AAs, respectively. Furthermore, expression of eIF-4E in bovine mammary tissues at different physiological periods was investigated by Northern blot analysis, using the cloned cDNA as the probe. eIF-4E was not detectable at prepubertal period and expressed at a very low level at the third estrous cycle. In the lactating mammary tissues, eIF-4E was highly expressed. Differential expression of eIF-4E in bovine mammary gland at distinct physiological stages indicates its potential involvement in mammary development. / Cell proliferation and apoptosis were also studied in the Escherichia coli (E. coli)-infected bovine mammary glands in the last study. Both proliferation and apoptosis increased in the mastitic tissue, as determined by immunohistological assays. Compared to the controls, expression of the pro-apoptotic proteins, Bax and interleukin-1beta converting enzyme (ICE), increased at 24 h and 72 h post-infection, whereas expression of the anti-apoptotic protein Bcl-2 decreased only at 24 h post-infusion. Induction of extracellular matrix (ECM)-degrading enzymes, including matrix metal loproteinase-9 (MMP-9), stromelysin-1 (SL-1) and urokinase-type plasminogen activator (uPA), was also observed in the mastitic tissue. Therefore, apoptosis may be mediated through pathways involving the actions of Bcl-2, Bax and ICE, and may partially be accounted by ECM breakdown. / Taken together, our study has demonstrated the effect of eIF-4E on bovine mammary cell proliferation. In addition, its involvement in bovine mammary gland development has been suggested. Finally, increased mammary cell apoptosis and proliferation during E. coli-induced mastitis has been revealed, in association with altered expression of apoptosis-related genes and ECM-degrading enzymes. Understanding the regulation of mammary cell proliferation and death may eventually lead to improvement of milk production.

No description available.

Bovine mammary glands respond to infection by foreign pathogens such as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) through changes in gene expression. Monitoring the gene expression profiles will contribute to better understanding of the pathology of mastitis, and provide important selective markers for future animal breeding programs. Using cultured bovine mammary duct epithelial cells and somatic cells from infected bovine mammary glands, this study first examined the existence of Toll Like Receptors in these two systems. In cultured duct epithelial cells stimulated with E. coli LPS, both TLR 4 and 2 mRNA up regulation was detected at 2h-72h and 12h-48h respectively. For S. aureus LTA TLR 2 mRNA was up regulated at 48 and 72h whereas for TLR 4 mRNA expression up regulation was detected at 24, 48, and 72h in comparison to the Oh (p<0.05). In the case of PGN, an abundant structural component of S. aureus, the expression of TLR 2 mRNA was significant (p<0.05) at 72h whereas TLR 4 mRNA expression increased at 24, 48, and 72h. The expression of these receptors was also monitored in milk cells from cows infected with either E. coli or S. aureus. However, results obtained from the milk cells were inconclusive due to the high individual variability. Afterwards, differential gene expression profiles were monitored by the Differential Display Polymerase Chain Reaction technique in the cultured duct epithelial cells in response to E. coli and S. aureus structural components. A total of 6 candidate fragments were identified for E. coli LPS induction, whereas only one fragment was identified for S. aureus LTA induction. After LTA induction, a specific band was found to be up regulated and confirmed to be GCP-2, a chemokine involved in neutrophil recruitment. In contrast, PGN induction resulted in no change in GCP-2 levels. In different preparations of cultured duct epithelial cells both GCP-2 and IL-8 were confirmed by real time PCR to be up regulated by LTA with a significance of (p<0.01) when compared to the control cells. In the case of the E. coli identified bands, a different approach is necessary to potentially confirm the origin of these fragments. Further large scale screening of the GCP-2 and IL-8 genes in dairy cattle is necessary to test for their potential use as targets to differentiate the mastitis resistant from the mastitis prone cows.

No description available.

No description available.

Although the occurrence of ketosis is a postpartum phenomenon, recent studies have focused on the prepartum period as key in the development of the disorder. Indicators of prepartum energy status, such as depressed dry matter intake (DMI) and elevated plasma non-esterified fatty acid (NEFA) concentrations have been associated with the occurrence of ketosis. The objective of this study was to investigate the role of phosphoenolpyruvate carboxykinase (PEPCK) in the periparturient and ketotic cow. The enzyme PEPCK catalyzes the rate limiting step in gluconeogenesis in hepatocytes. Whereas, in adipocytes, it has been suggested that PEPCK functions in the synthesis of glycerol for the formation of triacylglycerol (TAG) when plasma glucose concentrations are low. Thirty-four pregnant multiparous Holstein dairy cows were fed a single prepartum ration that consisted of 50% oat hay, 18% corn silage and 32% grain mix (DM basis). The ration was formulated to meet or exceed NRC requirements of 14% CP and 1.6 Mcal/kg NE[subscript L]. At calving, cows were transitioned onto one of two postpartum diets: control (n=14) or 3.5% supplemental fat (n=20). The postpartum diets, fed from wk 1 to 3, were formulated to isonitrogenous and to meet NRC requirements. Both diets consisted of 25% alfalfa, 25% corn silage and 50% grain mix. The control and fat diets contained 17.2 and 17.6% CP and 1.67 and 1.74 Mcal/kg NE[subscript L] respectively. Liver biopsies from 28 cows and adipose tissue biopsies from 6 cows were collected at -14, 2 or 3 and 14 d relative to calving. Tissue samples were analyzed for PEPCK mRNA and activity. All results were analyzed by period: prepartum (-21 to -2 d), freshening (2 to 7 d) and postpartum (8 to 21 d). In a previous study in our lab, 25 and 75% cows on the control and fat diets, respectively, experienced ketosis. In the current study there a 40% occurrence of ketosis for both control and fat diet groups. The high occurrence in both diets may be attributed to the rapid transition from the dry cow ration (70:30 forage to concentrate ratio, DM basis) to the lactating cow ration (50:50 forage to concentrate ratio, DM basis). The cows on the fat diet had lower serum glucose at freshening. Cows with ketosis had higher prepartum body weights (788 kg) than non-ketotic cows (743 kg; P<.1). No prepartum differences were seen in body condition score, DMI, NE[subscript L] balance, NEFA, glucose or ��-hydroxybutyrate concentrations were detected between ketotic and non-ketotic cows. Expression of adipose PEPCK mRNA was not different between ketotic and non-ketotic cows. However, hepatic PEPCK mRNA expression was higher in non-ketotic cows at freshening when compared to ketotic cows. Cows that experienced ketosis had lower hepatic PEPCK activity prepartum (6.6 vs. 9.3 units /min/g protein) and postpartum (7.6 vs. 10.2 units/min/g protein; P<0.5) when compared to non-ketotic cows. Our data indicated that hepatic PEPCK is a useful prepartum predictor of a cows susceptibility to ketosis. / Graduation date: 1998

The objective of the first part of this study was to observe and describe the profile of test-day somatic cell counts throughout a lactation in individual cows, and to examine the correspondence between such profiles and various lactation measures of cell count presently available. The objective of the second part of the study was to obtain estimates of the genetic and phenotypic parameters of lactation measures of cell count in different lactations, possibly for use in a breeding program. / In the first part of the study, the lactation cell count profiles of eighteen Holstein heifers of the Macdonald College Herd were individually examined. Weekly cell counts were expressed as deviations from the herd test-day average and plotted against week of test. A labile cell activity was found for most of the plotted lactations. The cellular responsiveness of cows facing external challenges seemed a trait peculiar to each individual. Lactation measures of cell count were unable to give good descriptions of cell count profiles of individual cows. However, they were better expressions of the lactation cell count performance than single test-day observations. / In the second part of the study, monthly cell count observations were obtained between February, 1977, and February, 1982, for the Holstein cows in herds enrolled on the official option of the Quebec Dairy Herd Analysis Service. Maximum likelihood, I-MINQUE (iterative Minimum Norm Quadratic Unbiased Estimation), and multivariate REML (Restricted Maximum Likelihood) procedures were used for the estimation of genetic and phenotypic parameters. / Repeatabilities of log test-day cell counts and log of test-day cell counts corrected for milk yield varied between 0.36 and 0.42 in first, second, third, fourth, and fifth and later lactations. Repeatabilities of test-day cell counts (cells/ml) in the five lactation groups varied between 0.17 and 0.25. Repeatabilities of lactation expressions for cell count between lactations ranged from 0.13 to 0.44. / Heritabilities of lactation expressions of cell count were low and varied from 0.06 to 0.14 in the five lactation groups examined; however, the genetic correlations between lactations were very close to unity, 0.90 to 0.97.