Tolerability of N-chlorotaurine in the bovine mammary gland

N-Chlorotaurine (NCT) is a promising endogenous agent for topical treatment of infections. We tested the tolerability and pharmakokinetics of NCT in the bovine mammary glands in a phase 1 study. Three concentrations of NCT in water (0.1%, 1.0%, 2.0%) were administered intramammarily in each of two cows. Into two quarters of the udder 100 ml NCT was injected into each twice daily for 5 d, while 0.9% NaCl was injected into the other two quarters in a randomized and blinded manner. Samples of milk were taken to determine the number of leucocytes and the activity of NCT, and samples of urine and blood to determine the taurine and chloride concentration. Chloride concentrations in serum samples were determined by an ISE-Unit of a Modular-System of the Roche Diagnostics company. The udder was monitored clinically for signs of inflammation. Oxidative activity could be detected in the milk after single irrigations for 15 min (0.1% NCT) and for maximally 5 h (1% and 2% NCT), respectively. On day 2, leucocytes increased to 4 x 10(6)/ml in the NCT group, while they remained 1 x 10(6)/ml in the saline group. However, on days 3-5 they increased to (5-7) x 10(6) in both the NCT and control group without any statistical difference. One day after the end of dosing the number decreased significantly and reached the baseline (<1 x 10(6)/ml) on day 10. The decrease was similar in both groups. Except for sporadic slight induration of single quarters in both groups and slight reduction of milk performance no disorders occurred. Taurine levels in blood and urine did not change. Irrigation of the bovine mammary gland with both NCT and saline caused a transient increase of leucocytes in the milk, but no severe side effects. The absence of residues and decay products may be a great advantage of NCT over other antimicrobial agents.     

Expression and regulation of glucose transporters in the bovine mammary gland

Glucose is the primary precursor for the synthesis of lactose, which controls milk volume by maintaining the osmolarity of milk. Glucose uptake in the mammary gland plays a key role in milk production. Glucose transport across the plasma membranes of mammalian cells is carried out by 2 distinct processes: facilitative transport, mediated by a family of facilitative glucose transporters (GLUT); and sodium-dependent transport, mediated by the Na+/glucose cotransporters (SGLT). Transport kinetic studies indicate that glucose transport across the plasma membrane of the lactating bovine mammary epithelial cell has a K(m) value of 8.29 mM for 3-O-methyl-D-glucose and can be inhibited by both cytochalasin-B and phloretin, indicating a facilitative transport process. This is consistent with the observation that in the lactating bovine mammary gland, GLUT1 is the predominant glucose transporter. However, the bovine lactating mammary gland also expresses GLUT3, GLUT4, GLUT5, GLUT8, GLUT12, and sodium-dependent SGLT1 and SGLT2 at different levels. Studies of protein expression and cellular and subcellular localizations of these transporters are needed to address their physiological functions in the mammary gland. From late pregnancy to early lactation, expression of GLUT1, GLUT8, GLUT12, SGLT1, and SGLT2 mRNA increases from at least 5-fold to several hundred-fold, suggesting that these transporters may be regulated by lactogenic hormones and have roles in milk synthesis. The GLUT1 protein is detected in lactating mammary epithelial cells. Its expression level decreases from early to late lactation stages and becomes barely detectable in the nonlactating gland. Both GLUT1 mRNA and protein levels in the lactating mammary gland are not significantly affected by exogenous bovine growth hormone, and, in addition, GLUT1 mRNA does not appear to be affected by leptin.     

Local immunostimulation of the bovine mammary gland with interleukin-2

One day after drying off, miniosmotic pumps releasing interleukin-2 were implanted into teat cisterns of six Jersey cows. Each cow had two quarters with (sensitized) and without (unsensitized) chronic Staphylococcus aureus infection. At 1 and 3 wk after implantation, three cows were sacrificed and mammary tissues processed to examine the cytological response to interleukin-2. Concentrations of lymphoid cells were determined in tissues lining teat and gland cistern linings, and plasma cells were characterized by Ig class. For total lymphoid cell and IgG1 and IgG2 plasma cell concentrations, sensitized placebo quarters and unsensitized interleukin-2 quarters were similar, indicating interleukin-2 stimulated a cellular immune response equal to that of quarters infected with S. aureus. Greatest response was observed in sensitized interleukin-2 quarters, suggesting that the cytokine was more effective in quarters with prior exposure to S. aureus antigens. Greater numbers of lymphocytes and macrophages were found in secretions of interleukin-2 quarters than in secretion of control quarters. Local interleukin-2 immunostimulation may supplement immunization procedures and enhance protection against natural S. aureus challenge.     

Lactoferrin concentration during involution of the bovine mammary gland.

Electroimmunodiffusion assay was used to quantitate changes in lactoferrin concentration in mammary secretions during involution of the bovine mammary gland. Concentration of lactoferrin began to increase 2 to 4 days after cessation of regular milking and continued to increase linearly at a rate of 1.15 mg/ml per day as a result of increased net synthesis of lactoferrin during the first 14 to 21 days of involution. Maximum lactoferrin concentration (approximately 20 mg/ml) was attained after 3 to 4 wk of involution. These changes represent a 100-fold increase in lactoferrin concentration over that in normal milk. Maximum lactoferrin concentration was variable between cows. In some cows, the concentration of lactoferrin plateaued at less than 10 mg/ml after 10 days of involution. In others, much higher lactoferrin concentrations of 75 to 100 mg/ml were measured. Lactoferrin concentration decreased markedly prior to parturition and onset of lactation. The increase in lactoferrin concentration during mammary gland involution appeared to be related closely to the process of involution.     

Prevalence and ultrastructural characteristics of bovine mammary corpora amylacea during the lactation cycle

Morphologic and histochemical study of spherical, lamellated inclusion bodies in bovine mammary parenchyma established presence of corpora amylacea. The majority (90%) were in alveolar lumina, followed by stroma (7%) and epithelium (3%). Occurrence of corpora amylacea was unrelated to lactation age, intramammary infection status, milk somatic cell concentration, or milk production. Quantitative analysis demonstrated a progressive increase of prevalence of these structures from the early to later stages of lactation, followed by a return during the late dry period to concentrations at early lactation. Ultrastructural study revealed deeply basophilic, dense, lamellated deposits (70%) and less dense, fibrillar deposits (30%). Dense bodies stained partially or not at all for amyloid but stained for presence of calcium salts, whereas fibrillar bodies were amyloid positive and calcium negative. Corpora amylacea were most prevalent in fully differentiated parenchymal areas (47.1 bodies/unit tissue area of 2.4 X 10(6) micron 2), less numerous in intermediately differentiated areas (23.4), and sparse in nonsecretory areas (7.1). Morphologic relationships between deposits and parenchyma suggest that corpora amylacea suppress milk secretion and flow during late lactation in isolated areas by engorging luminal spaces and clogging small ducts, leading to milk stasis and involution.     

Growth of Corynebacterium bovis in mammary secretions during physiological transitions of the bovine mammary gland

An in vitro microassay was used to evaluate growth of five strains of Corynebacterium bovis in mammary secretions collected from quarters of five Holstein cows at 0, 14, and 28 d of involution, at parturition, and 14 d after parturition. Variation in growth among different strains of Corynebacterium bovis was observed. Corynebacterium bovis grew well in mammary secretions obtained at the last milking of lactation, at parturition, and 14 d after parturition. However, growth of four strains of Corynebacterium bovis in mammary secretions obtained at 14 and 28 d of involution was reduced significantly. In contrast, a streptomycin-resistant strain of Corynebacterium bovis grew well in mammary secretions obtained during involution. These data suggest that mammary secretions support growth of Corynebacterium bovis during lactation but inhibit growth during the nonlactating period. Inhibition of growth in secretions collected during the nonlactating period may be associated with the high rate of spontaneous elimination of Corynebacterium bovis intramammary infection from cessation of milking to parturition. Conversely, enhanced growth in milk may be related to persistent Corynebacterium bovis intramammary infections during lactation.     

Histological response of the bovine mammary gland to intramammary devices.

Histologic response of the bovine mammary gland to presence of three intramammary device models (abraded, star, or grooved) was studied in 12 lactating cows. Uninfected quarters fitted with devices exhibited greater leukocyte infiltration into teat and gland cistem linings as well as into mammary parenchyma adjacent to the gland cistem compared with unfitted control quarters. Cytologic alterations to cistem linings in device-fitted quarters ranged from degeneration and sloughing of surface cells of the double-layered epithelium to hyperplasia, stratification, and keratin formation. In uninfected quarters, quantification of mammary parenchymal components showed no differences among models for percentages of epithelium, but percentage of lumen was lowest and stroma highest for the star intramammary device model, suggesting reduced secretory activity or mammary involution in response to these intramammary devices. Presence of bacterial infection amplified the histologic responses to all devices. Leukocyte infiltration remained greater in device-fitted quarters compared with controls but was elevated over uninfected quarters for all treatments. Likewise, hyperplasia, stratification, and keratin formation of the cistem epithelial lining were more frequently observed in infected quarters. Among models in infected quarters, percentage of lumen was lowest and stroma highest in quarters fitted with abraded devices. In most cases, presence of infection masked any effect of devices on mammary parenchymal components. Plaque formation was observed on all models and tended to be thicker on those retrieved from infected quarters. Electron microscopic examination showed that plaque was composed of leukocytes, cell debris, keratin, and amorphous material. Results demonstrated that most intramammary device models were successful in stimulating leukocytosis into the gland, and tissues from fitted quarters displayed alterations to cisternal linings; however, quarters fitted with these devices exhibited reduced secretory activity.     

Activation of immune cells in bovine mammary gland secretions by zymosan-treated bovine serum

Mastitis, caused by bacterial infection of the mammary gland, is a major disease of dairy cattle. The greatest risks of intramammary infection occur at the end of lactation and at the initiation of the next lactation when the cow calves. Treating serum with zymosan (yeast cell wall preparation) causes the complement to cleave, allowing this serum to serve as a source of complement fragment 5a (C5a), a potent chemoattractant and activator of the immune system. Our hypothesis was that intramammary infusion of zymosan-treated serum (ZTS) would recruit polymorphonuclear neutrophils (PMN) and generate prolonged activity in lymphocytes within the mammary gland. Ultimately this could help prevent bacterial infections in cows at dry-off and at the initiation of lactation. Two ipsilateral quarters of the mammary gland of each cow were infused with ZTS (12.5 mL/quarter), and 2 contralateral quarters were infused with saline in 8 cows shortly after lactation ended. Mammary secretions were collected periodically throughout the dry period and the first 2 wk of the next lactation. Activation status of lymphocytes and PMN in those secretions was assessed based on the intracellular presence or absence of IFN-γ and IL-8 as determined by flow cytometry. The ZTS infusion greatly increased PMN numbers in mammary secretions for the first week only. The percentage of IFN-γ positive lymphocytes and PMN, and the percentage of IL-8 positive PMN, exhibited a sustained increase in secretions from ZTS-treated quarters through the first 2 wk of lactation. The ZTS can stimulate PMN and lymphocyte-mediated immune defense mechanisms in the mammary gland, which may provide a useful means of preventing new intramammary infections during the dry period as well as at the initiation of lactation.     

Isolation of functionally active acini from bovine mammary gland.

Conditions to obtain high yields of intact acini from lactating bovine mammary glands and certain structural and functional characteristics of isolated acini were investigated. A two-factor experiment with three collagenase concentrations (100, 150, and 200 mg/100 ml) and incubation periods (40, 60, and 90 min) demonstrated that increases in both factors significantly increased net acini yield. Largest amounts of acini obtained, based on content of deoxyribonucleic acid, were 10.3% of the original tissue. Morphologically, fractions consisted primarily of acini or large cell clumps, and nearly all cells excluded trypan blue. Acini cultured in complete nutrient medium incorporated radioactive leucine into proteins. When acini were incubated in medium without supplemental amino acids, specific activity of synthesized proteins was correlated negatively with incubation time. During pulse labeling with radioactive L-leucine over 16 min, true labeling of acinar proteins occurred after 4 min. Sequential kinetics of pulse-chase labeling demonstrated a response pattern unique to the in vitro acinar system. Acinar protein synthesis was inhibited by cycloheximide and strongly stimulated by by 3',5'-cyclic adenosine monophosphate.     

Purification and properties of alkaline phosphatase in the lactating bovine mammary gland.

Alkaline phosphatase has been purified 1400-fold from homogenates of lactating bovine mammary tissue. The purification procedure included subcellular fractionation, solubilization with butanol, fractionation with acetone, chromatography on concanavalin A-Sepharose, DEAE cellulose, DEAE-Sephadex, and gel filtration on Sephadex G-200. The enzyme activity was measured with the substrate p-nitrophenylphosphate in three buffers, and the maximum rate occurred at pH 10. For maximum activity, Mg2+ was required. Substrate specificity studies at three pH values indicated that the enzyme had broad specificity. It catalyzed the hydrolysis of aliphatic and aromatic phosphates and pyrophosphates, but the phosphoprotein beta-casein was a poor substrate. Potent inhibitors of the enzyme were levamisole and sulfhydryl reagents (2-mercaptoethanol, dithiothreitol, and cysteine).     

Symposium review: The influences of heat stress on bovine mammary gland function

Heat stress reduces cow milk yield and results in a significant economic loss for the dairy industry. During lactation, heat stress lowers milk production by 25 to 40% with half of the decrease in milk synthesis resulting from the reduced feed intake. In vitro studies indicate that primary bovine mammary epithelial cells display greater rates of programmed cell death when exposed to high ambient temperatures, which may lead to a decrease in the total number of mammary epithelial cells in the mammary gland, partially explaining the lower milk production of lactating cows under heat stress. The function of mammary cells is also altered by heat stress. In response to heat stress, mammary cells display higher gene expression of heat shock proteins, indicating a need for cytoprotection from protein aggregation and degradation. Further, heat stress results in increased gene expression without altering protein expression of mammary epithelial cell junction proteins, and does not substantially influence the integrity of mammary epithelium. These data suggest that the mammary gland strives to maintain cell-to-cell junction integrity by synthesizing more proteins to compensate for protein losses induced by heat stress. During the dry period, heat stress negatively affects mammary gland development by reducing mammary cell proliferation before parturition, resulting in a dramatic decrease in milk production in the subsequent lactation. In addition to mammary growth, the mammary gland of the heat-stressed dry cow has reduced protein expression of autophagic proteins in the early dry period, suggesting heat stress influences mammary involution. Emerging evidence also indicates that heifers born to cows that experience late-gestation heat stress have lower milk yield during their first lactation, implying that the maternal environment may alter mammary gland development of the offspring. It is not clear if this is due to a direct epigenetic modification of prenatal mammary gland development by maternal heat stress. More research is needed to elucidate the effect of heat stress on mammary gland development and function.