Antibacterial activity of bovine mammary gland lymphocytes following treatment with interleukin-2.

The antibacterial characteristics of bovine mammary gland lymphocytes were evaluated following in vitro activation with recombinant human interleukin-2. Mammary gland effector cell populations were prepared by enzymatic digestion of mammary tissue followed by centrifugation through a discontinuous Percoll gradient. Functional activity of cells cultured for 48 h with or without interleukin-2 were evaluated by direct cytotoxicity using K562 target cells and by antibacterial activity against Staphylococcus aureus. Cytotoxic activity of interleukin-2-treated mammary gland lymphocytes was enhanced compared with control cultures, which exhibited no cytotoxic capabilities following 48 h of culture. Bactericidal activity also was elevated in cells cultured in interleukin-2-supplemented media. Enhanced expression of major histocompatability complex class II molecules on the surface of interleukin-2-treated lymphocytes was associated with the higher level of cell activation. Phenotypic analyses of the recovered population showed that the majority of cells were lymphocytes bearing CD2 molecules. It may be possible to activate these effector cell populations in vivo and increase resistance of the mammary gland to bacterial infections in a nonspecific manner.     

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.     

Relationship between milk lactoferrin and etiological agent in the mastitic bovine mammary gland

Bovine mastitis is one of the most deleterious diseases for dairy herds and is mainly caused by contagious and environmental bacterial pathogens. Among contagious bacteria, Staphylococcus aureus is the most prevalent, whereas the main environmental mastitis pathogens are Streptococcus uberis and Escherichia coli. Bovine lactoferrin (bLF) is an approximately 80-kDa glycoprotein present in milk that participates in the innate response of the mammary gland against bacterial infection. The objectives of the current study were to analyze potential changes in bLF milk concentration, which would constitute a response of the mammary gland toward mastitis induced by different etiologic agents, and to evaluate a possible relation between this response and pathogen susceptibility to bLF. Microbiology analysis and bLF quantification in milk from different bovine mammary gland quarters were performed. Infected quarters presented greater concentrations of bLF compared with those from microbiologically negative quarters. Analysis of individual pathogen contributions showed that most of this increase was attributable to Strep. uberis intra-mammary infection. The ability of mammary gland cells to synthesize bLF in response to Strep. uberis challenge was demonstrated by immunodetection of the protein in in vitro infection experiments. Susceptibility of Strep. uberis, E. coli, and Staph. aureus to the antimicrobial activity of bLF was determined by growth inhibition assays conducted with 4 different isolates of each species. Whereas Staph. aureus and E. coli were shown to be susceptible to this protein, Strep. uberis appeared to be resistant to the antimicrobial activity of bLF. Molecular typing of the 4 Strep. uberis isolates used throughout this study showed that this result was representative of the species and not exclusive of a particular strain. Results presented herein suggest that different bacteria species may elicit different mammary gland responses mediated by bLF secretion and that Strep. uberis has probably adapted to this immune reaction by developing resistance to bLF inhibitory action.     

Leptin affects prolactin action on milk protein and fat synthesis in the bovine mammary gland

Leptin, a protein hormone produced and secreted predominantly by white adipose tissue, has a critical role in the regulation and coordination of energy metabolism. Identification of leptin in the milk of several mammals, including humans, led us to investigate its presence and regulatory effect in the cow mammary gland. The expression of leptin receptor in tissue culture of lactating mammary gland was augmented approximately 25 times by prolactin, but had no effect on virgin calf mammary tissue. Expression of leptin in tissue culture from mammary glands of lactating cows was enhanced 2.2-fold by prolactin. No effect of prolactin on leptin and leptin receptor expression was found in mammary gland tissue culture from calves. Leptin-enhanced fatty acid synthesis in the presence of prolactin, but had no effect without presence of prolactin. A similar pattern was found in the expression of alpha-casein and beta-lactoglobulin in mammary gland explants from a lactating cow. Our findings indicate that leptin plays an important role in mammary gland lactogenesis, and that the expression of leptin requires the presence of prolactin.     

Culture of mammary epithelial cells from bovine milk.

Mammary epithelial cells are exfoliated into cows' milk and comprise a component of the somatic cells enumerated for milk quality control. This paper described the finding that these epithelial cells can be recovered from milk and grown in culture. The cultured cells were characterized for growth rate and growth potential, morphology by light and electron microscopy, presence of esterases and cytokeratins, and sensitivity to storage at 4 degrees C. Cultured mammary epithelial cells from milk may be useful to dairy scientists and mammary gland biologists.     

Effects of sodium fertilizers and supplements on milk production and mammary gland health

A series of experiments was conducted to investigate the effects of sodium fertilizers and supplements on the milk production and mammary health of dairy cows. In Expt 1, where sodium fertilizer was applied to productive pastures consisting mainly of the natrophile perennial rye-grass, the herbage sodium content and the milk yield of cows was increased and milk somatic cell count (SCC) reduced. In Expt 2, which used pastures containing less productive, natrophobic grasses and broad-leaved plants in Estonia, sodium fertilizer did not increase herbage sodium content and did not affect milk production or composition. In Expt 3 the sodium content of the diet of individually tethered cows was increased from 1 to either 6 or 11 g/kg dry matter (DM) by adding salt to their restricted feed allowance, and the cows' milk yield was increased by the high level of sodium supplement and milk SCC were reduced by both levels of sodium supplement. The calcium and magnesium status of cows was improved by the sodium supplement. In Expt 4 a low level of supplementary salt was included in the ration of tethered cows to increase the sodium content of the diet from 2 to 3.6 g/kg DM. No effects on milk yield or SCC were found, but the sodium supplement reduced Staphylococcus aureus contamination of the milk, but not the proportion of milk samples infected with Escherichia coli. It was concluded that the optimum dietary sodium concentration for maximum milk yield was greater than the published requirements, and that substantial increases in sodium intake above current requirements also reduced milk SCC.     

Membranes of mammary gland. XI. Marker enzyme distribution profiles for membranous components from bovine mammary gland.

Enzyme distribution profiles of clarified bovine mammary homogenates separated by equilibrium centrifugation on linear sucrose gradients suggested that several of the commonly utilized marker enzymes for rat liver are also valid markers for mammary cellular components. These marker enzymes include: Succinate dehydrogenase (mitochondria), nicotinamide adenine dinucleotide phosphate cytochrome c reductase and, to a lesser extent, retenone insensitive nicotinamide adenine dinucleotide cytochrome c reductase (endoplasmic reticulum), galactosyl transferase (Golgi apparatus), 5'-nucleotidase (plasma membranes), uric acid oxidase (microbodies), and acid phosphatase (lysosomes). Rotenone sensitive nicotinamide adenine dinucleotide cytochrome c reductase and sodium, potassium, magnesium-stimulated adenosine triphosphatase were widely distributed among subcellular fractions and are not valid marker enzymes. The boyant densities determined for the above fractions should aid in design of methods to obtain enriched sources of these components for analysis.     

Natural protective factors in bovine mammary secretions following different methods of milk cessation

Bovine mammary secretions were obtained during late lactation and early involution to determine if different methods of milk cessation influenced milk yield, composition, and in vitro growth of coliform mastitis pathogens. Cows (n = 8/group) producing about 13 kg of milk prior to experimentation were dried off by abrupt or intermittent milk cessation. An additional group was dried off by intermittent milk cessation and fed only hay during the last week of lactation. Cows milked intermittently produced significantly less milk during the last week of lactation than cows dried off by abrupt milk cessation. Mammary secretions from cows milked intermittently and fed only hay contained higher concentrations of somatic cells, lactoferrin, immunoglobulin G, and bovine serum albumin, a lower citrate:lactoferrin molar ratio, and were more inhibitory to in vitro growth of Escherichia coli and Klebsiella pneumoniae throughout most of the experimental period than mammary secretions from cows dried off by intermittent or abrupt milk cessation. Few differences in mammary secretion composition or in vitro growth of mastitis pathogens were observed between cows dried off by intermittent or abrupt milk cessation. Data suggest that growth of mastitis pathogens in mammary secretions may be related to natural protective factors, which can be manipulated by different methods of milk cessation.     

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.     

Distribution of milk in the goat mammary gland and its relation to the rate and control of milk secretion

Changes with time in the distribution of milk between different storage regions of the mammary gland were studied in goats. Milk was allowed to drain from the gland through a catheter (catheter milk); the gland was then milked by hand, before (hand milk) and after the administration of oxytocin (alveolar or residual milk). The hand-milk fraction was of negligible volume. There was an essentially linear increase in the catheter-plus hand-milk fraction from 0 to 16 h. The alveolar fraction increased up to 6 h, but thereafter remained constant to 16 h. There was considerable but consistent variation between animals in the alveolar fraction as a proportion of total milk volume. The proportion of residual milk was inversely related to milk secretory rate per unit weight of mammary tissue. The hourly administration of oxytocin without milking had no apparent effect on the distribution of milk in the gland 1 h later.     

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.     

Effects of photoperiod modulation and melatonin feeding around drying-off on bovine mammary gland involution

The risk for a dairy cow to acquire new intramammary infections is high during the transition from lactation to the dry period, because of udder engorgement and altered immune functions. Once the gland is fully involuted, it becomes much more resistant to intramammary infections. Therefore, strategies to depress milk yield before drying-off and accelerate the involution process after drying-off could be beneficial for udder health. The objective of this study was to assess the effect of photoperiod manipulation and melatonin feeding from 14 d before to 14 d after drying-off on the speed of the involution process. Thirty Holstein cows in late lactation were randomly allocated to one of the following treatments: (1) a long-day photoperiod (16 h of light: 8 h of darkness), (2) a short-day photoperiod (8 h of light: 16 h of darkness), and (3) a long-day photoperiod supplemented by melatonin feeding (4 mg/kg of body weight). Milk and blood samples were collected on d ?26, ?19, ?12, ?5, ?1, 1, 3, 5, 7, 10, and 14 relative to the last milking to determine concentrations of mammary gland involution markers and serum prolactin. Additional blood samples were taken around milking on d ?15, before the start of the treatments, and on d ?1, before drying-off, to evaluate the treatment effects on milking-induced prolactin release. The short-day photoperiod slightly decreased milk production and basal prolactin secretion during the dry period. The milking-induced prolactin surge was smaller on d ?1 than on d ?15 regardless of the treatments. Lactoferrin concentration, somatic cell count, and BSA concentration as well as matrix metalloproteinase-2 and -9 activities increased in mammary secretions during the first 2 wk of the dry period, whereas milk citrate concentration and the citrate:lactoferrin molar ratio decreased. The rates of change of these parameters were not significantly affected by the treatments. The long-day photoperiod supplemented by melatonin feeding did not affect milk production, prolactin secretion, or mammary gland involution. Under the conditions in this study, photoperiod modulation and melatonin feeding did not appear to affect the rate of mammary gland involution.     

Purification and properties of an acid phosphatase from lactating bovine mammary gland

An acid phosphatase was partially purified from the cytosol of lactating bovine mammary gland by precipitation with ammonium sulfate and protamine, chromatography on carboxymethyl cellulose, and gel filtration on Sephadex G-75. The enzyme hydrolyzed aromatic phosphates but was less active toward alkyl phosphates, ATP, and phosphoproteins (casein and phosvitin). A sulfhydryl group seems to be essential for activity, since dithiothreitol and cysteine activated the enzyme; compounds that react with the sulphydryl groups in proteins were inhibitory. Orthovanadate, phosphate, and zinc ions also inhibited the phosphatase.