Growth of Staphylococcus aureus and Streptococcus species in bovine mammary secretions during the nonlactating and peripartum periods following intramammary infusion of lipopolysaccharide at cessation of milking.

The objective was to determine if induced mammary inflammation at cessation of milking influenced growth of gram-positive mastitis pathogens in mammary secretions, particularly during early involution. Growth of all mastitis pathogens evaluated was similar in cell-free fat-free mammary secretions from LPS-infused and control glands. These data indicate that intramammary infusion of LPS at cessation of milking did not alter growth of gram-positive mastitis pathogens in mammary secretion during the nonlactating period. Stage of lactation and the nonlactating period influenced bacterial growth and marked differences between bacteria and among strains of a bacterial species were observed. Staphylococcus aureus grew well in secretions collected during late lactation, but growth decreased during early- and mid-involution and increased again in secretions obtained near parturition. Streptococcus agalactiae and Strep. uberis grew better in mammary secretion obtained during involution than in secretions collected during late or early lactation. Streptococcus dysgalactiae grew well in mammary secretions at all time periods. These data demonstrate the variability of mastitis pathogen growth during physiologic transitions of the bovine udder.     

Growth inhibition of Escherichia coli and Klebsiella pneumoniae during involution of the bovine mammary gland: relation to secretion composition

Mammary secretions from 12 Holstein dairy cows were collected to evaluate growth inhibition of Escherichia coli and Klebsiella pneumoniae during involution and during physiologic transitions of the mammary gland. Mammary secretions obtained during late lactation poorly inhibited growth of E coli and K pneumoniae. However, as involution progressed, mammary secretions increasingly inhibited growth of both coliform mastitis pathogens. Greatest inhibition of E coli and K pneumoniae growth was observed when mammary glands were fully involuted. Growth inhibition remained high until 7 days before parturition, and then it decreased significantly (P less than 0.05) to that observed during late lactation. Inhibition of coliform mastitis pathogen growth was associated with high concentrations of lactoferrin and immunoglobulin G, decreased citrate concentration, and a low citrate to lactoferrin molar ratio. These data suggested that differences in susceptibility or resistance to new intramammary infection with coliform mastitis pathogens during the nonlactating period may be attributable, in part, to marked changes in mammary secretion composition that develop during physiologic transitions of the mammary gland. Resistance of the fully involuted mammary gland to coliform infection may be associated with high concentrations of natural protective factors.     

Effects of intra-mammary bacterial infection with coagulase negative staphylococci and stage of lactation on shedding of epithelial cells and infiltration of leukocytes into milk: comparison among cows, goats and sheep

The effects of mammary gland bacterial infection and stage of lactation on leukocyte infiltration into the mammary gland were compared among cows, goats and sheep. Animals were at two stages of lactation: mid or late. In mid-lactation animals, bacterial-free glands and coagulase negative Staphylococcus (CNS)-infected glands were compared. In late lactation only uninfected glands were studied. Of mid-lactation bacteria-free animals, goats had the highest number of leukocytes and % polymorphonuclears (PMNs), whereas sheep had the lowest and leukocytes number in cows were intermediate between sheep and goats. Based on %PMN, two cell clusters were found in sheep, which overlapped with the parallel cell clusters of cows and goats, but with a slightly higher number of leukocytes in each cell cluster. At late lactation, goats had higher values for %PMN and leukocyte numbers in comparison to cows, which had a similar cellular profile to sheep. The cellular immune response to CNS infection was similar for the three animal species, although the number of cells was different, while the basal cell level at mid-lactation and especially at the end of lactation was species specific.     

Involution and redevelopment of the mammary gland in goats. Histological-histochemical studies after a lactation time of 8-10 months

7 chamois-coloured mountain goats were used to investigate histologically and histochemically the processes of involution and redevelopment of the mammary gland after a lactation period of 8 to 10 months. Tissue specimens were obtained by incision biopsy at drying-off two months prepartum and afterwards in intervals of 8-16 days up to parturition. The findings of this investigation were compared with results of two former investigations, in which involution and proliferation of the gland were studied separately, thus precluding an overlapping of the two processes. At drying-off, after an 8 to 10 month-lactation, sections indicative of active lactation occurred concomitantly with sections at various stages of involution, as well as early stages of redeveloping alveoli. At 16 days post drying-off, the sections indicating lactation at first examination were in a stage of maximum involution and transformation. Thus, the time required for involution was reduced by half compared to drying-off at peak lactation. At 32 days post drying-off, stages of proliferation predominated with only a few involuting glands. Specimens obtained thereafter contained only redeveloping glands. The histological and histochemical differentiation between areas of involution and those of proliferation may present difficulties during the mid-portion of the dry period. There appears to be an association between the length of the time interval from drying-off to parturition and the rate of tissue transformation in the caprine mammary gland; the rate is increased when the duration of the dry period is reduced.     

Growth inhibition of environmental mastitis pathogens during physiologic transitions of the bovine mammary gland

Ten dairy cows were infused intramammarily near drying off with concanavalin A (conA) or phytohemagglutinin (PHA). Mammary secretions were collected during physiologic transitions of the udder and were used in an in vitro microbiological assay to determine growth inhibition of mastitis pathogens. As mammary involution progressed, in vitro growth inhibition of Klebsiella pneumoniae, Escherichia coli, and Streptococcus uberis increased. Mammary secretions from conA- and PHA-treated glands had significantly increased bacterial growth inhibition. Secretions contained significantly increased concentrations of lactoferrin and a decreased citrate:lactoferrin molar ratio earlier in the dry period than did control mammary secretions. Greatest bacterial growth inhibition was observed in mammary secretions obtained 7 days before parturition. However, differences in secretion composition or bacterial growth inhibition were not found between conA- or PHA-treated and control udder halves during the prepartum period. Bacterial growth inhibition by mammary secretion decreased markedly during early lactation. A highly significant positive correlation was found between bacterial growth inhibition and concentrations of lactoferrin, serum albumin, and immunoglobulin G. A highly significant negative correlation was found in the citrate:lactoferrin molar ratio during early involution and the peripartum period.     

Modifications of the defense and remodeling functionalities of bovine neutrophils inside the mammary gland of milk stasis cows received a commercial dry-cow treatment

An appropriate length of milk stasis between two consecutive lactations of dairy cows is crucial for sustainable milk production. This dry period of cows allows extensive remodeling and sufficient cell renewal in mammary gland. Nevertheless, early dry period is one of the most risky stages in cow lactation cycle to intramammary infection. Dry-cow treatment through teats is, therefore, widely practiced at the commencement of milk stasis. Neutrophils are the most abundant cellular components in cow mammary secretion during early dry period, which in turn attribute to the meanwhile elevation of somatic cell counts and matrix metalloproteinase-9 (MMP-9) level. This study used bovine peripheral neutrophils as a cell model to examine the mode of modifications in their defense and remodeling functionalities after infiltration into mammary gland during early dry period. Results indicate a dose-dependent suppression of phorbol 12-myristate 13-acetate-stimulated free radical production and induction of MMP 9 degranulation in bovine peripheral neutrophils exposure to the d 7-dry secretion of cows received dry cow treatment at d 0 in milk stasis. Meanwhile, an enhancement of plasminogen activation and TNF-α shedding on bovine peripheral neutrophils were also observed. These two cellular events might be involved in the functional modifications on infiltrated neutrophils during early dry period. In conclusion, the opposite trend of modifications in the defense and matrix remodeling functionalities of neutrophils inside the mammary gland of cows at early dry period reflect the collaboration of infiltrated neutrophils for promoting extensive glandular remodeling at minimum compromise of local defense during the acute involution period without apparent disturbance by dry cow treatment.     

饲喂高精料日粮对泌乳期山羊泌乳性能及抗氧化能力的影响

为了研究长期饲喂高精料日粮对泌乳期山羊泌乳性能和乳腺组织抗氧化能力的影响,实验选用12只健康经产泌乳中期关中奶山羊,随机分为两组,分别饲喂精粗比为35∶65(低精料组, LC, n=6)和65∶35(高精料组, HC, n=6)的日粮。实验期共10周,实验期间采集羊奶与血液;实验结束后采用静脉注射的方法对山羊进行安乐死,随后采集乳腺组织并迅速置于液氮中速冻后保存于-70 ℃待测。结果显示,HC组山羊乳产量显著高于LC组(P<0.01),同时乳中乳糖,乳蛋白的比例随着泌乳时间的增加而显著升高。与LC组山羊比较,HC组山羊乳腺组织中SREBP1和GLUT1 mRNA表达显著下降(P<0.05);磷酸化AKT1蛋白表达呈下降趋势(P=0.08);除此之外,HC组山羊乳腺组织的总抗氧化能力T-AOC(P=0.06)和GSH酶活均呈下降趋势(P=0.08)。这些结果表明,长期饲喂高精料日粮可降低乳腺组织的抗氧化能力,同时可降低乳中乳脂率以及乳腺组织中SREBP-1和P-AKT的表达。     

Lysosomal enzymes in bovine mammary leukocytes during the nonlactating period

Leukocytes from mammary secretions in dairy cows were collected during the nonlactating and postpartum periods. Differential cell counts, viability and activity of peroxidase, N-acetyl-beta-D-glucosaminidase (NAGase, beta-glucuronidase and alpha-mannosidase in cells were determined. Cell viability (trypan blue exclusion) was 75-80% during most of the nonlactating period, but declined to 45-50% by parturition. Polymorphonuclear neutrophils (PMN) predominated during the first week of involution, after which macrophages were the predominant cell type. Peroxidase activity in leukocytes from mammary secretions was high in early involution, probably reflecting the predominant peroxidase-containing PMN. Peroxidase activity declined through the remaining nonlactating and postpartum periods. The activity of NAGase was variable in early involution, then increased to a peak during the mid-nonlactating period, before declining prior to parturition. Activity of beta-glucuronidase generally was unchanged during the nonlactating period, although NAGase and beta-glucuronidase activities were significantly and positively correlated throughout the period studied. Activity of alpha-mannosidase changed in a manner similar to peroxidase activity.     

Quantitation of calmodulin in the mammary gland of the lactating sow

It has been suggested that calmodulin, a calcium-binding protein, has a functional role during milk secretion. High levels of calmodulin are present during lactation in rat mammary glands and a substantial increase has been observed in the bovine mammary gland prior to parturition. In the sow, regressed glands involute while suckled glands remain highly active even though they are under the same hormonal influence. In this study, tissue samples were taken from suckled and regressed glands of the same sow at both peak and late lactation. Calmodulin and total protein were measured in tissue homogenate supernatants. Residual milk was apparent in regressed glands during mid lactation but not in the same glands by late lactation. Calmodulin levels in tissue were the same for both suckled and regressed glands. There was a slight but non-significant increase in the tissue calmodulin level from peak to late lactation. Protein levels declined significantly from mid to the late stage of lactation. There was no change in protein level between the suckled and regressed glands. Calmodulin may be responsible for casein phosphorylation and/or the mediation of prolactin action on the gland. The precise regulatory mechanisms relating hormonal control to calmodulin levels during lactation need further investigation.     

Oxytocin concentrations of cattle in response to milking stimuli through lactation and mammary involution II

Patterns of oxytocin release to milking stimuli over a lactation and during mammary involution, were examined in seven Holstein cows used in the previous study. Blood samples were taken before, during and after milking or udder massage. Oxytocin as measured by radioimmunoassay increased within O to 2 min after attachment of the milking unit. Oxytocin levels fluctuated during milking and declined after the initial increase. Oxytocin often dropped below basal levels after milking. Milking-induced oxytocin release decreased as lactation advanced. The maximal increment for oxytocin release was significantly different between early and late lactation. The time taken to reach peak hormone concentrations declined across lactation. Relative amounts of oxytocin released in response to milking stimuli were significantly more in early than during late lactation. Cows released oxytocin during mammary involution with relatively large, rapid increases to udder massage. A distinct peak was observed and return to basal concentrations was rapid. The mean increment of oxytocin concentration above basal was 51.6 ±10.1 uU/ml. Maximal oxytocin levels occurred 1.6 ±.2 min (0 to 2 min) after initial stimulation. The total amount of oxytocin released in response to stimulation was 1.2 ±.1 uU/ml. In summary, a continuous or multiple release of oxytocin occurs during milking. The sensitivity of the neuroendocrine reflex for oxytocin appears dynamic. Changes in maximal concentrations and total amounts of hormone released in response to milking during lactation, and the relationship between these variables and basal concentrations suggest a gradual loss of sensitivity from the early stages of lactation to mammary involution.     

Comparison of morphology, viability, and function between blood and milk neutrophils from peak lactating goats

The morphological features of blood and milk neutrophils from peak lactating goats were compared using light microscopy, scanning electron microscopy and flow cytometry in order to investigate the cytological changes of neutrophils after migration into the mammary gland. The kinetics of reactive oxygen intermediates (ROI) generation and gelatinase release of blood and milk neutrophils, with or without stimulation of phorbol 12-myristate, 13-acetate ester (PMA), were used to characterize their responses to inflammatory stimuli. Neutrophils isolated from goat milk were highly segmented and contained multi-lobed nuclei. Ultrastructurally, milk neutrophils were more ruffled on the surface compared to blood neutrophils. Approximately 30% of milk neutrophils were undergoing cell death, either necrosis or apoptosis, in contrast to 8% of blood neutrophils. The ROI production of activated milk neutrophils peaked earlier than blood neutrophils, but the duration and the intensity were much less. Neutrophils from both sources augmented the release of gelatinase in response to PMA (1 ng/mL). However, the amount of gelatinase released from milk neutrophils was lower (P < 0.05) than that of blood neutrophils. In summary, more neutrophils become apoptotic and necrotic in the mammary gland, presumably due to spontaneous aging, the process of diapedesis, and the interaction with milk components. Milk neutrophils have impaired functionalities in comparison with blood neutrophils. The information is relevant when studying mammary gland immunity and related diseases, such as mastitis.     

荷斯坦奶牛乳腺发育中的乳糖变化研究

目的:研究荷斯坦奶牛乳腺发育中乳糖的变化规律.方法:高效液相色谱法测定荷斯坦奶牛乳腺发育中乳糖的含量.结果:围产期,乳腺中才出现可检测到的乳糖;泌乳期乳糖含量增高,泌乳140d达到峰值;退化期乳腺乳糖含量迅速降低,退化30d含量很少.结论:不同生理时期,荷斯坦奶牛乳腺中的乳糖含量不同.     

Immunocyte populations in the mammary gland of the rat at different stages of pregnancy and lactation.

Immunocyte populations of apparently normal rat mammary glands were examined at different stages of lactation using immunofluorescence techniques. IgA-and IgG-containing cells were present before parturition; IgA-containing cells were numerous than IgG- or IgM-containing cells during the entire period from pregnancy through parturition and lactation to involution.     

Modulation of bovine mammary neutrophil function during the periparturient period following in vitro exposure to recombinant bovine interferon gamma

Effects of recombinant bovine interferon (rBoIFN) gamma on mammary gland neutrophil activity during the periparturient period were studied. Bovine mammary gland neutrophils were isolated and incubated in mammary gland secretions obtained from Holstein-Friesian cattle during the last 2 weeks of gestation. Cell functions were evaluated following treatment with 10 U, 100 U, and 1000 U of rBoIFN-gamma. Bacterial phagocytosis, bactericidal activity and chemiluminescence were significantly lower for neutrophils incubated in mammary gland secretions when compared with control neutrophils incubated in Hank's balanced salt solution. Treatment of mammary neutrophils with rBoIFN-gamma reversed the suppressive effects of mammary secretions resulting in higher chemiluminescent activity and significantly more bacterial phagocytosis and bactericidal activity when compared with untreated controls. Results from these preliminary in vitro data suggest that rBoIFN-gamma therapy may modulate mammary gland neutrophil functions in vivo and possibly facilitate the rapid clearance of mastitis-causing pathogens mammary glands during the periparturient period.     

Course of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) in mammary secretions of the goat during end-pregnancy and early lactation

The epidermal growth factor (EGF) plays a crucial role in mammogenesis in many species. In ruminants, studies are limited, as EGF does not occur in peripheral plasma and specific analytical systems do not exist. Therefore a heterologous radioimmunoassay based on rhEGF was set up to monitor EGF in mammary gland secretions from goats during end-pregnancy and early lactation. IGF-I was measured with an established radioimmunoassay. Samples were collected from 13 goats for 25 days ante-partum and 25 days post-partum. Mammary gland secretions were obtained ante-partum by removing a small amount of the udder secretions (control half) or milking (stimulated half). Post-partum normal milk samples were collected. Blood samples were drawn by jugular venipuncture for the same period. EGF was found to occur in different molecular weight forms in the mammary glands. For routine measurements these proteins were extracted with acetone and not further separated. IGF-I and EGF concentrations in mammary secretions and similarly IGF-I in blood were high ante-partum and decreased slightly towards birth. IGF-I but not EGF is found in the peripheral plasma. Whereas IGF-I concentrations in blood were quite constant post-partum, IGF-I and EGF dropped in mammary secretions close to the detection limits. The decrease was more pronounced in the stimulated half than in the control half. The data support a synergistic role for EGF and IGF-I for mammogenesis. Both factors are further influenced by the milking stimulus and thus the functional state of the udder.     

BCL-2 family of proteins and mammary cellular fate

This review focuses on the pro-apoptotic and anti-apoptotic Bcl-2 family members involved in apoptosis, which is the predominant process controlling cell remodelling during post-lactational mammary gland involution. The members of the Bcl-2 protein family, whose expression levels are under the control of lactogenic hormones, internally control this mechanism also during lactation. They can physically interact with each other, sometimes in an antagonistic manner. Mammary glands undergo repeated cycles of structural development, functional differentiation and regression, therefore provide a unique model for investigating this family of proteins that regulate the fate of the secretory cells and consequently milk yield. The involvement of Bcl-2 family members is reviewed in mammary tissue during morphogenesis, at different stages of lactation cycle and in comparison with dairy and laboratory animals.     

Frequency of isolation of environmental mastitis-causing pathogens and incidence of new intramammary infection during the nonlactating period

Quarter samples (n = 6,328) of mammary secretions were collected from 160 cows during physiologic transitions of the udder to determine the frequency of isolation of mastitis-causing pathogens and the incidence of new intramammary infections (IMI) during the nonlactating period. None of the cows in the herd was infected with Streptococcus agalactiae, and the prevalence of Staphylococcus aureus was low. Cows were not treated with antibiotics at cessation of milking. A threefold increase in the percentage of quarters infected with major mastitis-causing pathogens developed from late lactation to early involution. Coliforms and streptococci other than Str agalactiae accounted for 94% of major pathogen infections. The number of quarters infected with coagulase-negative staphylococci increased slightly from late lactation to early involution, whereas the number of quarters infected with Corynebacterium bovis decreased markedly. Major pathogens caused 101 of 153 IMI at parturition and greater than 90% were caused by streptococci and coliforms. At parturition, 51 of 52 minor pathogen IMI were caused by coagulase-negative staphylococci. During early lactation, there was a marked decrease in quarters infected with major pathogens; however, the number of quarters with major pathogen IMI during early lactation was 2.3 times higher than the number of quarters infected before cessation of milking. The number of quarters with minor pathogen IMI during early lactation was the same as at parturition, but a marked decrease in quarters infected with coagulase-negative staphylococci and a marked increase in C bovis IMI developed from parturition to early lactation.     

Apoptotic Cell Death, bax and bcl-2 Expression During Sheep Mammary Gland Involution

Involution in ovine mammary tissue was studied by light and electron microscopy, and bax and bcl-2 protein distribution was examined by immunohistochemistry from the last day of lactation until the 8th day of drying off. The mammary gland alveoli were examined and the area of glandular epithelium was evaluated morphometrically. Regression of mammary gland epithelium by apoptosis was first identified 2 days after the end of lactation, and increased until day 8. Bax protein was detected throughout this period and was highest on the eighth day. A weak positive reaction for bcl-2 was observed only on days 1 and 8 after cessation of lactation. It is concluded that sheep mammary gland involution involves cell death by apoptosis and that bcl-2 gene family members are involved in the process.     

Protein restriction in pregnant‐ and lactating rats‐induced oxidative stress and hypohomocysteinaemia in their offspring

Hyperhomocyteinaemia has been associated with pathological and stressful conditions and considered as a risk factor for cardiovascular diseases. Maternal protein restriction during late pregnancy and postnatal period is a stressful state to their offspring which is associated with disruption of various physiological processes. The objective of the current study was to evaluate the effects of maternal protein restriction during late pregnancy and early postnatal periods on systemic oxidative stress parameters and on homocysteine metabolism of their offspring. For this purpose, 12 female rats were divided into two groups: a control group (C) with free access to standard diet (20% protein) and a protein restricted group (PR) with free access to a low protein diet (7% protein) from the day 14 of pregnancy until day 14 after delivery. An increase of thiobarbituric acid‐reactive substance levels (TBARS) in plasma (+40%, p < 0.01) and in liver (+37%, p < 0.001) with a concomitant decrease in the activities of catalase (CAT) and superoxide dismutase (SOD) observed in undernourished pups suggest the possibility of oxidative stress. Moreover, no significant changes in total glutathione levels (tGSH) were also observed to be associated with a significant decrease in homocysteine concentrations (?35%; p < 0.001). In their mothers, an alteration of antioxidant enzyme activities (CAT and SOD) and slight increase of TBARS levels were observed. Whereas homocysteine and tGSH levels were unchanged in comparison with those fed ad libitum. These results suggested that PR diet given to mothers during late pregnancy and early postnatal periods induced oxidative stress and hypohomocysteinaemia in their offspring.     

A study of bovine T-cell subsets in the blood and mammary gland secretions during the dry period

Blood and mammary secretions were obtained from cows throughout the dry period. Quantitative and qualitative assays were performed to determine the cell types and cell distributions at weekly intervals from day of dry off until parturition. The total cell counts in secretions increased during involution and remained at high levels until a few weeks prepartum. The macrophages were the predominant cell type in mammary secretions whereas the numbers of lymphocytes were always less than neutrophils or macrophages. Enriched mononuclear cell populations derived from blood and mammary secretions were also evaluated using "T-cell rosette" assays. Changes observed in the relative distribution of three T-cell subsets in secretions did not reflect the dynamics of the cells in the peripheral blood. T-cell subsets that predominated in mammary secretions were the EN+ EAET+ and EN-EAET+ phenotypes. Distinct patterns of migration or differentiation of T-cell subsets were suggested by the changes of subsets observed in mammary secretions collected throughout the dry period.