Adrenomedullin (AM) and adrenomedullin binding protein (AM-BP) in the bovine mammary gland and milk: Effects of stage of lactation and experimental intramammary E. coli infection

Adrenomedullin (AM) has been characterized as an endogenous tissue survival factor and modulator of many inflammatory processes. Because of the increased susceptibility of the mammary gland to infection during the time surrounding parturition in the cow, we investigated how milk and tissue content of AM and its binding protein (AM-BP) might be affected by the stage of lactation and the udder health status. Milk and mammary biopsy samples were obtained from Holstein cows 21 days prior to and at various times after calving to represent the dry period and early and mid-stages of lactation. Additional cows received an intramammary challenge with Escherichia coli for immunohistochemical characterization of AM and AM-BP. Milk AM concentrations were relatively constant across the stages of lactation while AM-BP increased two-fold (P<0.04) between early and mid-lactation. Milk AM (P<0.04) and AM-BP (P<0.03) increased as somatic cell counts (SCCs) increased within a given stage of lactation. Tissue content of both (AM and AM-BP) were significantly affected by stage of lactation, lowest in the dry period and progressively increasing to peak at mid-lactation as well as increasing in association with higher levels of SCCs. Following E. coli challenge, AM increased in epithelial cells surrounding mammary alveoli presenting high levels of SCCs. The data suggest that AM and AM-BP are cooperatively regulated in the mammary gland during lactation; changes in localized tissue AM and AM-BP content reflect a dynamic regulation of these tissue factors in the bovine mammary gland consistent with their protective effects within inflamed tissue.     

Lactation persistency: insights from mammary cell proliferation studies

A persistent lactation is dependent on maintaining the number and activity of milk secreting cells with advancing lactation. When dairy cows are milked twice daily, the increase in milk yield from parturition to peak lactation is due to increased secretory activity per cell rather than to accretion of additional epithelial cells. After peak lactation, declining milk yield is due to loss of mammary epithelial cells by apoptosis. During lactation, only 0.3% of mammary cells proliferate in a 24-h period. Yet this proliferative rate is sufficient to replace most mammary epithelial cells by the end of lactation. Management practices can influence lactation persistency. Administration of bovine somatotropin may enhance persistency by increasing cell proliferation and turnover, or by reducing the rate of apoptosis. Increased photoperiod may also increase persistency of lactation by mechanisms that are as yet undefined. Increased milking frequency during the first weeks of lactation increases milk yield, even after return to less frequent milking, with increases of approximately 8% over the entire lactation. A mammary cell proliferation response to frequent milking during early lactation appears to be involved. Conversely, advanced pregnancy, infrequent milking, and mastitis increase death of epithelial cells by apoptosis. Regulation of mammary cell renewal provides a key to increasing persistency. Investigations to characterize epithelial cells that serve as the proliferative population in the bovine mammary gland have been initiated. Epithelial cells that stain lightly in histological sections are evident through all phases of mammary development and secretion and account for nearly all proliferation in the prepubertal gland. Characterization of these cells may provide a means to regulate mammary cell proliferation and thus to enhance persistency, reduce the effects of mastitis, and decrease the necessity for a dry period.     

Evaluation of L-selectin expression and assessment of protein tyrosine phosphorylation in bovine polymorphonuclear neutrophil leukocytes around parturition

Impaired polymorphonuclear neutrophil leukocyte (PMN) function around parturition has been associated with increased clinical mastitis in dairy cows. Rolling and attachment of PMN to the endothelium is the first step in the recruitment process and is accomplished by interaction between L-selectin on PMN and its ligand on endothelial cells. Furthermore, tyrosine phosphorylation is involved in the initiation of many PMN functions. The objective of this work was to determine changes in expression of L-selectin and tyrosine phosphorylation in the perinatal period. Eight clinically healthy Holstein cows were used as PMN donors at d-21, -14, -7,0 (calving), +1, +2, +7, +14, +28. Evaluation of L-selectin expression was carried out on activated and resting PMN. Anti-bovine L-selectin monoclonal antibody (MAB) and flow cytometric analysis were used to measure the percentage of PMN fluorescing and receptor expression (log mean fluorescent channel, LMFC). Activated and resting PMN showed similar trends in % PMN fluorescence and LM FC. The percentage of PMN fluorescing tended to decrease at parturition, followed by a significant increase at d +14 and +28 (P < 0.02). For LMFC a decrease was observed on d +1 followed by an increase through d +28 (P < 0.01). Protein tyrosine phosphorylation of lysates prepared from PMN isolated throughout the study was detected by electrophoresis and western blotting using anti-phosphotyrosine MAB. Several protein bands were tyrosine phosphorylated. Two of these bands (42-44 kDa and 90 kDa) varied in intensity over time. The intensity of the 42-44 kDa band gradually increased from d -7, peaked at d +7 (P < 0.03), and steadily decreased to d +28 (P < 0.02). Antibody to activated mitogen protein kinase reacted with the 42-44 kDa band. Reduced PMN function during the periparturient period could be related to reduced L-selectin adhesion molecules on the cell surface, and to modulation in the phosphorylation of functionally important molecules.     

Morphometric analysis of involuting bovine mammary tissue after 21 or 42 days on non-suckling

Mammary gland involution was morphologically evaluated 21 or 42 d after prevention of suckling of one udder half in 10 crossbred beef cows. Parenchymal tissue was taken from lower, middle and upper zones of each quarter from the teat to the ventral body wall. Udder halves, trimmed of extraparenchymal tissue, were weighed and used for DNA determination. DNA content was reduced 50 and 64% after 21 and 42 d of involution. However, the percentage of tissue occupied by epithelium was similar in suckled and nonsuckled glands. Well-differentiated cells, typical of suckled glands, were rarely observed in nonsuckled glands. Alveolar structure was evident in nonsuckled glands, but the number of cells per alveolar cross-section was reduced (30 vs 22). Unlike in suckled glands, there was a marked gradation in classification of epithelial cells across zones in involuting glands. For example, nearly 10% of the epithelium was well-differentiated in the tissue from the upper zone, whereas no well-differentiated cells were found in the lower zones. Regression of the mammary parenchyma does not occur uniformly through the udder, so use of single biopsy to study involution should be avoided. Presence of alveoli after 42 d indicates that redevelopment of the udder with subsequent lactations is less dramatic than suggested from study of other species.     

Effects of stress on endocrine and metabolic processes and redirection: cross talk between subcellular compartments

Recent advances in genome analysis and biochemical pathway mapping have advanced our understanding of how biological systems have evolved over time. Protein and DNA marker comparisons suggest that several of these systems are both ancient in origin but highly conserved into today's evolved species. However, remnants of some of the more ancient functions of these chemical systems can run in conflict with the functions that those same pathways serve in complex organisms and tissue systems today. Relevant to the present topic, nitric oxide (NO) and superoxide anion (O₂^•−), ancient cellular molecules in evolutionary terms, are recognized today as both necessary for the well-being and stable health of cells but also injurious to cells as elaborated in conjunction with the cellular stress response. Why the dichotomy? This question underlies one of the basic issues challenging researchers as well as practitioners in their approach to disease management. The fundamental proinflammatory response of the innate immune system of the host is needed for pathogen control but can be injurious to tissues from “collateral damage” from NO- and O₂^•−-derived reactive molecules capable of affecting protein function via post-translational chemical modification. This review highlights newer aspects of the biochemistry of the NO- and O₂^•−-mediated innate proinflammatory response and further show how protein and tissue damage via overproduction of reactive nitrogen and oxygen intermediary molecules such as peroxynitrite (ONOO⁻) might be targeted to specific epitopes of proteins. Changes in the regulation of metabolism in response to proinflammatory disease states are discussed for GH signal transduction and tissue specificity.     

Comparison of acetate tape impression with squeezing versus skin scraping for the diagnosis of canine demodicosis

This study compared the sensitivity of acetate tape impression and skin squeezing with that of deep skin scraping for the diagnosis of demodicosis in dogs. Demodex canis was detected in 100% of acetate tape impressions obtained after skin squeezing and in 90% of deep skin scrapings. There was a significant difference (P < 0.001) between the techniques in the total number of mites detected. Acetate tape impression with skin squeezing was found to be more sensitive than deep skin scraping and is an alternative diagnostic method for canine demodicosis.     

Mammary histology and alveolar cell differentiation during late gestation and early lactation in mammary tissue of beef and dairy heifers

Quantitative histology of mammary parenchymal tissue from 16 Hereford and 16 Holstein heifers was determined for tissue obtained on day 150, 180, and 280 of gestation and on day 49 of lactation. Percent area occupied by stromal tissue was progressively decreased on each consecutive sample day during gestation in Herefords and was lower in both breeds during lactation. Overall, area occupied by stromal tissue elements was also greater in Herefords. Percent lumenal space and number of cells per alveolar cross section was consistently greater for Holsteins and increased across sample periods in both breeds. During lactation more than 40% of the alveolar cells in Herefords were characterized as poorly differentiated, but in Holstein heifers nearly all of the cells were classified as either intermediate or fully differentiated. These data provide additional evidence to support the idea that much of the difference in milk production between beef and dairy animals depends on increased mammary function. Whether differences in milk production within dairy breeds are also explained by altered cellular differentiation remains to be determined.     

REVIEW ARTICLE: An integrated view on how the management of the dry period length of lactating cows could affect mammary biology and defence

The dry period is necessary to facilitate cell turnover in the bovine mammary gland and to optimize milk production in the next lactation. An 8‐week dry period has long been the golden standard of management for dairy cows. Genetic improvements and new management technologies have led to higher milk production and a need for re‐evaluation of the dry period length. Over the last decade, dry period length has been proposed to be shortened or eliminated mainly from an economic point of view. However, the influence of modified dry period length on the immune defence of the bovine mammary gland and the occurrence of new intramammary infections has not yet been appreciated. The objective of this review is to discuss the bovine mammary gland biology, defence and systemic health when the dry period length is modified. Shortening or eliminating the dry period may minimize or remove the impact of milk accumulation at dry off, thereby lessening the immunodeficiency of the dam that is characteristic of this period. Composition of mammary secretions may change and the extent of tissue remodelling may be reduced when the dry period is reduced or eliminated. Additionally, impact of the dry period length on energy and nutritional status, and on hormonal and local regulatory factors, lead us to hypothesize that changing the dry period length might also affect the response to intramammary infection. It is concluded that there is a need to integrate mammary gland biology and defence mechanisms in studies dealing with modified dry period lengths.