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United States Department of Agriculture

Agricultural Research Service

2011 Annual Report

1a.Objectives (from AD-416)
The objectives of the research project plan are as follows: .
1)to identify disease resistance genes and immunological responses that influence the course of intramammary infection;.
2)to discover and evaluate effective biotherapeutics for the prevention and treatment of bovine mastitis; and.
3)to develop strategies that promote cell replacement in the bovine mammary gland.

1b.Approach (from AD-416)
To identify disease resistance genes that influence the course of intramammary infection (Objective.
1)we will: .
1)compare differential innate immune response patterns and host gene expression profiles that are elicited in response to intramammary pathogens that are readily cleared from the gland versus those that establish chronic infection;.
2)determine whether experimentally-induced inflammation enhances clearance of mastitis pathogens that cause chronic subclinical mastitis; and.
3)compare the inflammatory and gene expression responses of primiparous versus multiparous cows. To discover and evaluate effective biotherapeutics for the prevention and treatment of bovine mastitis (Objective.
2)we will: .
1)test the efficacy of intramammary infusion of recombinant bovine sCD14 as a means to recruit neutrophils and promote clearance of E. coli;.
2)test the effectiveness of the organic irritant dextran at dry-off to prevent new intramammary infections;.
3)evaluate the anti-inflammatory and microbicidal activity of bovine bactericidal-permeability increasing protein (BPI) in various biological fluids as an initial indicator of its utility in the treatment of intramammary and systemic infections; and.
4)evaluate the ability of cis-urocanic acid to inhibit neutrophil-induced respiratory burst activity and injury to the mammary epithelium. To identify strategies that promote cell replacement in the bovine mammary gland (Objective.
3)we will focus on the biology of bovine mammary stem cells, which are crucial for the proliferation replacement of mammary epithelial cells. In prepubertal heifers, we will: .
1)identify mammary stem cells by their ability to retain bromodeoxyuridine label for an extended time and develop genetic markers for these cells, by isolating them from tissue using laser microdissection, and performing microarray analysis to identify markers that distinguish stem cells from non-stem cells; and.
2)we will evaluate methods to promote expansion of the stem cell population in vivo by modulating key signaling pathways.

3.Progress Report
Additional studies are being completed to confirm and extend our finding that infusion of xanthosine into mammary glands of prepubertal heifers increases the number of mammary stem cells. (1) In vitro studies demonstrate that xanthosine induces symmetrical division of stem cells and expansion of the population. (2) Preliminary evaluation of an ongoing study indicates that xanthosine treatment of mammary glands of prepubertal heifers may alleviate the decreased milk production that occurs when heifers are fed to gain at an accelerated rate. Laser excision of putative mammary stem cells and control cells from histological sections led to identification of genes that are more highly expressed in mammary stem cells. Initial evaluation of these stem cell biomarkers is promising, as cells that express the markers are present in the appropriate location and abundance in mammary tissue sections. Increased knowledge of bovine mammary stem cells may permit altering their activity to promote the repair of mammary tissue that is damaged by mastitis and to enhance milk production efficiency. Reactive oxygen and nitrogen products are synthesized by immune and nonimmune cells in response to infection. Although production of these reactants is needed to destroy mastitis-causing pathogens, they can damage the milk secreting cells of the mammary gland. We characterized the distribution of these reactive compounds in response to experimentally induced intramammary infection. The production of reactive molecules was unevenly distributed within the tissue. Distribution patterns of the highly reactive peroxynitrate and resulting nitrated proteins coincided with areas of mitochondrial tissue damage. Strategies targeting the reactivity of peroxynitrite are being developed and will be a focus of our next project plan. Objectives for renewal of the current project plan include an evaluation of physiological and genetic factors that influence incidence and severity of mastitis. (1) We identified animals with differing physiological responses to injection of endotoxin that should equate with high and low susceptibility to mastitis. The initial trial to identify these animals used yearling Holstein heifers and their lifetime mastitis incidence will be monitored. (2) Genetic profiling is being used to identify animals that possess the above physiological responses. The populations will be used to evaluate the relationship between this genotype and mastitis susceptibility. Endophyte-infected fescue contains ergot alkaloids that inhibit prolactin secretion. This in turn is thought to reduce mammary differentiation and milk yield. Effect of feeding cows a diet containing endophyte-infected fescue, during the two months prior to calving and the first week after calving, are being evaluated. Although milk production was initially reduced in cows consuming endophyte-infected feed and in the positive controls (receiving injections of a purified ergot alkaloid), ultimately production of treated cows exceeded that of control cows. Additional laboratory analyses will provide insight into underlying physiological mechanisms and possible management options.

1. Ovariectomy alters prepubertal development of mammary myoepithelial cells (MC) but the mechanisms involved in the differentiation of mammary stem cells and progenitor cells to MC are not understood. We therefore analyzed the expression of MC differentiation markers in intact and ovariectomized heifers at 55, 70, 85, 100, 130 and 160 days of age. Our results showed that ovariectomy affects MC expression of myoepithelial cell markers as well as the pattern of MC development. Because MC are known to limit parenchymal growth in other species, our observations open new avenues for future studies on the regulation of prepubertal mammary development. A manuscript has been prepared and submitted for publication.

Review Publications
Farmer, C., Palin, M.F., Gilani, G.S., Weiler, H., Vignola, M., Choudhary, R.K., Capuco, A.V. 2010. Dietary genistein stimulates mammary development in gilts. Animal. 4(3):454-465.

Pezeshki, A., Capuco, A.V., De Spiegeleer, B., Peelman, L., Stevens, M., Collier, R.J., Burvenich, C. 2010. An integrated view on how the management of the dry period length of lactating cows could affect mammary biology and defence. Journal of Animal Physiology and Animal Nutrition. 94:7-30.

Choudhary, R.K., Daniels, K.M., Clover, C.M., Garrett, W.M., Capuco, A.V. 2010. A Rapid Method for BrdU Immunostaining in Bovine Mammary Cryosections that Retains RNA Quality. Journal of Dairy Science. 93:2574-2579.

Rinaldi, M., Li, R.W., Capuco, A.V. 2010. Mastitis associated transcriptomic disruptions in cattle. Veterinary Immunology and Immunopathology. 138(4):267-279.

Capuco, A.V., Akers, R.M. 2011. Galactopoiesis/Effects of hormones and growth factors. In: Fuquay, J.W., Fox, P.F., McSweeney, P.L.H, editors. Encyclopedia of Dairy Science. 2nd edition. San Diego, CA: Academic Press. p. 26-31.

Capuco, A.V., Akers, R.M. 2011. Galactopoiesis/Effects of bST. In: Fuquay, J.W., Fox, P.F., McSweeney, P.L.H., editors. Encyclopedia of Dairy Science. 2nd edition. San Diego, CA: Academic Press. p. 32-37.

Akers, R.M., Capuco, A.V. 2011. Lactogenesis. In: Fuquay, J.W., Fox, P.F., McSweeney, P.L.H., editors. Encyclopedia of Dairy Science. 2nd edition. Sandiego, CA: Academic Press. p. 15-19.

Capuco, A.V., Ellis, S. 2010. Mammary Glands: Developmental Changes. In: Pond, W.G., Bell, A.W., editors. Encyclopedia of Animal Science. 2nd edition. London, England: Taylor & Francis. p. 720-722.

Chase, C.C., Elsasser, T.H., Spicer, L.J., Riley, D.G., Lucy, M.C., Hammond, A.C., Olson, T.A., Coleman, S.W. 2011. Effect of growth hormone administration to mature miniature Brahman cattle treated with or without insulin on circulating concentrations of insulin-like growth factor-I and other metabolic hormones and metabolites. Domestic Animal Endocrinology. 41(1):1-13.

Foster, E.B., Fisher, G., Sartin, J.L., Elsasser, T.H., Wu, G., Cowan, W., Pascoe, D.D. 2011. Acute regulation of IGF-I by alterations in post-exercise macronutrients. Amino Acids. Available:

Elsasser, T.H., Kahl, S., Li, C. 2011. Modeling the effects of estradiol and progesterone on the acute phase proinflammatory axis: Variability in tumor necrosis factor-¿, nitric oxide, and xanthine oxidase responses to endotoxin challenge in steers. Domestic Animal Endocrinology. 40(4):213-221.

Last Modified: 4/24/2014
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