Effects of intramammary infusions of casein hydrolysate,ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid,and lactose at drying-off on mammary gland involution

The transition from the lactation to the dry period in dairy cows is a period of high risk for acquiring new intramammary infections. This risk is reduced when involution of mammary glands is completed. Consequently, strategies that accelerate the involution process after drying-off could reduce the incidence of mastitis. The objective of this study was to assess the effect of 3 different treatments on mammary gland involution. Each quarter of 8 Holstein cows in late lactation was randomly assigned at drying-off to an intramammary infusion of casein hydrolysate (CNH; 70 mg), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA; 5.7 g), lactose (5.1 g), or saline 0.9% (control) solutions. Milk samples were collected on the last 2 d before and 1, 3, 5, 7, 10, and 14 d after the last milking for determining concentrations of mammary gland involution markers. Lactoferrin, somatic cell counts (SCC), BSA, and Na⁺ concentrations, as well as matrix metalloproteinase-2 and matrix metalloproteinase-9 activities gradually increased in mammary secretions during the first 2 wk following the last milking, whereas milk citrate and K⁺ concentrations decreased. As involution advanced, the Na⁺:K⁺ ratio increased, whereas the citrate:lactoferrin ratio decreased. Compared with mammary secretions from control quarters, mammary secretions of quarters infused with CNH had higher SCC on d 1, 3, 5, and 7, and greater BSA concentrations on d 1, 3, and 5. Similarly, the CNH treatment induced a faster increase in lactoferrin concentrations, which were greater than in milk from control quarters on d 3, 5, and 7 after drying-off. Milk citrate concentrations were unaffected by CNH but the citrate:lactoferrin ratio was lower in CNH-treated quarters on d 3 and 5 than in control quarters. Moreover, CNH treatment hastened the increase in Na⁺ concentration and in the Na⁺:K⁺ ratio on d 1. Infusion of CNH also led to an increase in proteolytic activities, with greater matrix metalloproteinase 9 activities on d 1 and 3. The EGTA infusion increased SCC above that of control quarters on d 1 and 3 but it had no effect on the other parameters. Lactose infusion had no effect on any of the involution markers. In this study, intramammary infusions of CNH were the most efficient treatment to accelerate mammary gland involution, suggesting a potential role of CNH as a local milk secretion inhibitor during milk stasis.     

Intramammary inoculation of Panax ginseng extract in cows at drying off enhances early mammary involution

This study was designed to evaluate the effects of a single intramammary infusion of Panax ginseng extract on cell proliferation and death mechanism in bovine mammary gland during early involution. Eight mammary quarters from six non-pregnant cows in late lactation were infused with 10 ml of ginseng solution (3 mg/ml), six quarters were treated with 10 ml of placebo (vehicle alone) and six quarters were maintained as uninoculated controls. Milking was interrupted after infusion. Animals included in the three groups were slaughtered 7 d after inoculation and samples for histological analysis were taken. Morphometric analysis showed a significant increase in percentages of mammary tissue area occupied by stroma in ginseng-treated quarters compared with controls. A significant increase of immunostained area for bax protein and active caspase-3 was observed in ginseng-treated quarters compared with controls, whereas no differences were observed for bcl-2 immunostaining. Expression of bax mRNA was significantly higher in ginseng-treated quarters than in controls. The bax/bcl-2 ratio indicated a significant predominance of bax over bcl-2 mRNA expression in ginseng-treated quarters compared with controls. The rise of epithelial and stromal cell apoptosis in situ by TUNEL was more marked in quarters treated with ginseng than in controls. Ginseng inoculation had no effect on the number of epithelial and stromal proliferating cells labelled with Ki-67 antibody. Ratio of apoptotic to proliferating cells was higher in quarters treated with ginseng compared with controls, indicating a net loss of cells in parenchymal components. Also, the intramammary inoculation of ginseng extract at drying off increased the rate of mammary cell apoptosis without inhibiting cell proliferation. Taken together, these changes are indicative of mammary regression enhancement during early involution.     

Effect of a biological response modifier on cellular death mechanisms at drying off

Agents that increase natural protective mechanisms have been proposed for prevention and treatment of intramammary infections. The objective of this study was to describe the effects of a single intramammary infusion of a lipopolysaccharide (LPS)-based biological response modifier (BRM) on cellular death mechanism in uninfected and Staphylococcus aureus-infected bovine mammary glands during involution. Three groups of 12 cows, each one including 6 Staph. aureus-infected and 6 uninfected, were infused in two mammary quarters with BRM or placebo and slaughtered at 7, 14 and 21 d of involution. In infected quarters, BRM treatment produced a significant increase in percent of stained epithelial cells for the apoptosis-promoting protein Bax at every observation period. In addition, BRM produced a significant increase of immunostained stromal cells for Bax compared with placebo-treated quarters. BRM treatment produced an increase in percentages of epithelial cells staining with active caspase-3 at 7 d and 14 d of involution compared with placebo-treated quarters and a significant decrease in percentages of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive epithelial cells at 7 d and 21 d of involution. In addition, BRM treatment caused an increase in percentage of stromal cells immunostaining for active caspase-3 and TUNEL. An increase of active caspase-3 and TUNEL epithelial and stromal cell immunostaining was observed in Staph. aureus-infected compared with uninfected quarters. Cellular proliferation, determined by Ki-67 immunostaining, was increased in epithelial and stromal cells from Staph. aureus-infected compared with uninfected quarters at every observation period. These results provide new insights into the mechanism of mammary cell death in uninfected and Staph. aureus-infected bovine mammary gland during involution and illustrate the effects of LPS-based BRM on apoptosis and cell proliferation during mammary involution.     

Effects of yeast expressed recombinant interleukin-2 and interferon-gamma on physiological changes in bovine mammary glands and on bactericidal activity of neutrophils

The physiological effects of intramammary infusions of recombinant bovine cytokines in six lactating dairy cows on the quality and yield of milk and the bactericidal activity of milk neutrophils were investigated. Recombinant bovine interleukin-2 (rboIL-2) and interferon-gamma (rboIFN-gamma) were produced in the yeast Pichia pastoris. Two animals were given rboIL-2 (2 x 10(5) units) in two quarters, two animals were given rboIFN-gamma (6.5 x 10(5) units) in two quarters, and the other two cows received a dose of rboIL-2 in one quarter and rboIFN-gamma in a second quarter. In addition, each animal was given phosphate-buffered saline (PBS) in the other two quarters as a control. Somatic cell counts and conductivity of the fore milk were monitored before and after infusion. Neutrophils were isolated from quarter milk samples 36 h after infusion of cytokine or PBS and their bactericidal activities against Staphylococcus aureus were measured in vitro with a colorimetric assay. Quarters infused with rboIL-2 or rboIFN-gamma showed significant but transitory increases in both milk somatic cell counts and conductivity when compared with preinfusion values and with control quarters. There were minimal effects on daily milk yield. Neutrophils isolated from milk from quarters infused with rboIL-2 showed enhanced bactericidal activity against Staph. aureus. The bacterial killing from rboIL-2 treated quarters was significantly greater, with a mean of 63.5% compared with a mean of 5.4% for neutrophils taken from uninfected quarters to which PBS had been administered. The bactericidal activities for quarters treated with rboIFN-gamma and infected quarters treated with PBS were 15.0 and 30.0% respectively. The results indicate that intramammary infusions of rboIL-2 and rboIFN-gamma to lactating cows are well tolerated, and that rboIL-2 can activate milk neutrophils and augment their bactericidal activity.     

Influence of nonlactating and peripartum bovine mammary secretions on growth of Staphylococcus species

Bovine mammary secretions from individual quarters were collected from five cows at 0, 14, and 28 d of involution, at parturition, and 14 d after parturition and used in a microassay to evaluate growth of several Staphylococcus species. All Staphylococcus species evaluated followed similar growth patterns in secretions. Mammary secretions obtained at 14 and 28 d of involution were poor media for growth of Staphylococcus species. Conversely, mammary secretions collected at cessation of milking, parturition, and early lactation supported growth of all species evaluated. Growth of Staphylococcus species in mammary secretions differed slightly among cows but was similar among quarters of a cow. Results suggested that the ability of bovine mammary secretions to support or inhibit growth of Staphylococcus species was related to functional states of the mammary gland.     

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.     

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.     

Effect of intramammary infusion of chitosan hydrogels at drying-off on bovine mammary gland involution

The transition from lactation to the dry period in dairy cows is a period of high risk for acquiring new intramammary infections. This risk is reduced when the involution of the mammary gland is completed. Accordingly, approaches that speed up the involution process after drying-off could reduce the incidence of mastitis. The current study aimed to develop a biological response modifier that could be injected into cow teats to promote immune cell migration and speed up involution. Chitosan, a natural polysaccharide derived from chitin, is able to trigger host innate immunity. We developed 2 formulations made from either high- or low-viscosity chitosan. Both are liquid at room temperature but form a hydrogel at body temperature. In the first experiment, each udder quarter of 7 Holstein cows in late lactation was randomly assigned at drying-off to receive one of the following intramammary infusions: 2.5 or 5 mL of 5% (wt/vol) low-viscosity chitosan hydrogel, 5 mL of 5% high-viscosity chitosan hydrogel, or 5 mL of water. Milk (mammary secretion) samples were collected from each quarter on d ?4, ?1 (drying-off), 1, 3, 5, 7, and 10. Milk somatic cell counts and the concentrations of involution markers such as BSA, lactate dehydrogenase, and lactoferrin were measured in each sample. In comparison with the control, the chitosan hydrogel infusions significantly hastened the increases in somatic cell counts, BSA and lactoferrin concentrations, and lactate dehydrogenase activity in mammary secretions. No major differences between sources or volumes of chitosan were observed for the measured parameters. The compatibility of this approach with an internal teat sealant was verified in the second experiment. Each udder quarter of 8 Holstein cows was randomly assigned at drying-off to receive one of the following intramammary infusions: 5 mL of 2% low-viscosity chitosan hydrogel, 4 g of an internal teat sealant, a combination of sealant and chitosan, or 5 mL of water. Milk (mammary secretion) samples were collected from each quarter on d ?4, ?1 (drying-off), 5, and 10 to measure involution markers. These results suggest that chitosan hydrogel infusion hastened mammary gland involution and activate immune response, which may reduce the risk of acquiring new intramammary infections during the drying-off period. Those results were not affected by the presence of the teat sealant, showing that both approaches are fully compatible and could be used in combination.     

Effect of a biological response modifier on expression of growth factors and cellular proliferation at drying off

Agents that increase natural protective mechanisms have been proposed for the prevention and treatment of intramammary infections. Staphylococcus aureus is a major pathogen causing primarily subclinical chronic mastitis that responds poorly to antibiotic therapy. The objectives of this study were to describe the effects of a single intramammary infusion of a lipopolysaccharide-based biological response modifier (BRM) on mammary epithelial cellular proliferation and expression of insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor (VEGF) in uninfected and Staph. aureus-infected bovine mammary glands during involution. Three groups of 12 cows, 6 Staph. aureus-infected and 6 uninfected, were infused with BRM or placebo in 2 mammary quarters and killed at 7, 14, and 21 d of involution. The proportion of infected quarters, mammary cell proliferation, and IGF-I and VEGF expression were evaluated. Biological response modifier treatment decreased the proportion of Staph. aureus-infected mammary quarters at 7 d of involution, but a similar number of isolations were observed at 14 and 21 d of involution in either treated or control quarters. The percentage of proliferating mammary epithelial cells was higher in infected than uninfected quarters at every observation period, irrespective of the treatment administered, whereas uninfected BRM-treated quarters showed increased cell proliferation at 7 d of involution. Insulin-like growth factor-I expression in uninfected quarters was not affected by treatment and showed a decrease at 21 d of involution. Expression of IGF-I was greater in infected than uninfected quarters at every observation period, irrespective of the treatment received. Expression of VEGF was greater in BRM-treated uninfected quarters at 7 d of involution compared with controls. In infected quarters, VEGF expression was lowest in BRM-treated quarters at 7 d of involution and increased throughout the observation period. Conversely, untreated infected quarters showed the highest VEGF expression at 7 d and decreased at 21 d of involution. Mammary cell proliferation and expression of IGF-I and VEGF were increased in Staph. aureus-infected quarters. Increased mammary cell proliferation and VEGF expression were observed in BRM-treated quarters during the first week of involution.     

Mammary serum amyloid A3 activates involution of the mammary gland in dairy cows

The dry period is a nonlactating phase in which senescent mammary cells are regenerated, which is thought to optimize milk production in the subsequent lactation. In bovines, the dry period normally coexists with pregnancy and the lactogenic hormones delay mammary gland involution and impair the activation of immune system to fight the risk of intramammary infections. Conventionally, long dry periods of up to 60 d are required to allow sufficient mammary regeneration for full milk yield in the next lactation. The aim of this study was to evaluate the potential of mammary serum amyloid A3 (M-SAA3) as an activator of the involution of the mammary gland. One milligram of recombinant M-SAA3 and the corresponding negative controls (saline solution and lipopolysaccharide) were infused into the mammary gland via the teat canal, and mammary secretion samples were taken during the first 3 d after drying off to analyze metalloproteinase activity, somatic cell count, protein, and fat contents. Primary mammary gland epithelial cell cultures and bovine dendritic cells, obtained from necropsy tissue and blood, respectively, were incubated with and without M-SAA3 and cytokine expression was quantified. Last, the protective role of the M-SAA3 against infections was evaluated after a Staphylococcus aureus challenge. Matrix metalloproteinase 9 activity, a key protein that directly participates in the onset of the involution process, was greater in quarters treated with the M-SAA3. Protein content was increased in mammary secretions compared with control quarters. M-SAA3 increased cytokines directly related to innate immunity in both epithelial and dendritic cells and reduced the infection by Staphylococcus aureus.     

Intramammary infections in heifers during early lactation following intramammary infusion of pirlimycin hydrochloride or penicillin-novobiocin at the first milking after parturition

A study was conducted to determine whether intramammary antibiotic treatment of heifer mammary glands following the first milking after calving was effective for reducing the percentage of mammary quarters infected during early lactation. Jersey and Holstein heifers from two research herds were assigned to one of three treatment groups: (1) no intramammary infusion following the first milking after parturition, (2) intramammary infusion of all quarters with pirlimycin hydrochloride following the first milking after parturition and (3) intramammary infusion of all quarters with novobiocin sodium plus penicillin G procaine following the first milking after parturition. Almost 93% of Jersey heifers (40/43) and 73.1% of quarters (125/171) were infected at the first milking. Almost 77% of quarters (33/43) were cured following treatment with pirlimycin, 61.8% (21/34) were cured following treatment with penicillin-novobiocin and 39.6% (19/48) of infections were eliminated spontaneously in the untreated control group. Significantly fewer infections were observed in pirlimycin or penicillin-novobiocin treated mammary glands of Jersey heifers during early lactation than in untreated control mammary glands. Almost 89% of Holstein heifers (32/36) and 52.8% of quarters (76/144) were infected at the first milking. About 57% (12/21) of quarters were cured following treatment with pirlimycin, 41.4% (12/29) were cured following treatment with penicillin-novobiocin and 23.1% (6/26) of infections were eliminated spontaneously in the untreated negative control group. Significantly fewer infections were observed in pirlimycin treated mammary glands of Holstein heifers during early lactation than in untreated control mammary glands. However, no significant differences were observed following penicillin-novobiocin treatment of Holstein heifers after the first milking of lactation compared with untreated control quarters. Coagulase-negative staphylococci, Streptococcus uberis and Streptococcus dysgalactiae subsp dysgalactiae were isolated most frequently in heifers from both herds.     

Staphylococcus aureus mastitis: pathogenesis and treatment with bovine interleukin-1 beta and interleukin-2.

Polymorphonuclear leukocytes play a central role in the pathogenesis of bovine mastitis. Intramammary challenge with Staphylococcus aureus was shown to induce both quantitative and qualitative changes in mammary gland polymorphonuclear leukocytes. Intramammary infusion of recombinant bovine interleukin-1 beta and interleukin-2 elicited a similar cellular response. Staphylococcus aureus, interleukin-1 beta, and interleukin-2 all increased the number of somatic cells after intramammary infusion and activated the inducible superoxide production in milk polymorphonuclear leukocytes. Interleukin-2 also activated phagocytosis of these cells, and their activation was maintained for 3 to 5 d after intramammary administration. Interleukin-1 beta and interleukin-2 were moderately effective in the therapy of experimental S. aureus mastitis. Approximately 54% of the glands treated with interleukin-1 beta responded to therapy by transiently clearing the milk of S. aureus, 30% of which relapsed, and a total of 38% of the treated glands remained cured. In contrast, 83% of glands treated with interleukin-1 beta responded to therapy, but 50% of these quarters relapsed. A total of 42% of the quarters treated with interleukin-1 beta remained cured. Homologous recombinant cytokines are effective immunomodulators that augment natural defensive mechanisms similar to the normal response to pathogens and may prove to be suitable alternatives to, or may be used in combination with, antibiotics as effective mastitis therapeutic agents.     

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.     

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.     

Effects of bovine somatotropin on beta-casein mRNA levels in mammary tissue of lactating cows

Bovine somatotropin (bST) increases milk production in lactating cows through its effect on nutrient partition and maintenance of mammary cell function. A positive relationship between bST treatment and abundance of β-casein mRNA in mammary tissues from lactating cows was hypothesized. In mammary tissue isolated from 14 midlactation Holstein cows, β-casein mRNA was 35.4% higher among 7 cows receiving continuous bST infusions at 29 mg/d for 63 d compared with tissue from 7 untreated control cows. To investigate whether increased β-casein mRNA resulted from a direct effect of bST on the mammary gland, explants of mammary tissue from other lactating cows that had not received bST were incubated with bST and prolactin in 2 experiments. Mammary explant cultures taken from 2 lactating cows that had not been milked for 48 h were supplemented with either prolactin or bST. Both prolactin and bST stimulated higher levels of β-casein mRNA in the mammary explants compared with their non-supplemented counterparts. Explant cultures from 4 additional lactating cows were prepared from rear quarter mammary tissue subjected to milking intervals of 6 h for right rear quarters or 20 h for left rear quarters. Both bST- and prolactin-mediated increases in β-casein mRNA were dependent on milking intervals. That is, levels of β-casein mRNA were increased by bST or prolactin supplementation in explants isolated from the mammary quarters biopsied 20 h after milking but not for those biopsied at 6 h after milking. Results are consistent with a potential role for bST in up-regulating or sparing β-casein mRNA levels in lactating bovine mammary tissue in a manner similar to prolactin.     

Approaches to the manipulation of mammary involution

Mammary involution is a gradual process that occurs following cessation of milking. Regression of mammary secretory tissue accompanies dramatic changes in secretion composition during the transition from lactation to involution. Conversely, rapid differentiation of secretory tissue and copious accumulation of colostrum occur as parturition approaches. The duration of the nonlactating period, mammary gland health, and secretory cell response to hormones influence subsequent lactational performance in most species. Manipulation of the bovine mammary gland in an attempt to hasten involution has been studied. The primary objective of these studies was to determine if hastened involution would decrease new intramammary infections during the early nonlactating period. Results of these studies have also led to a more fundamental understanding of events that occur during physiological transition of the mammary gland. Adequate regression, proliferation, and differentiation of mammary secretory epithelium during the nonlactating period of ruminants appear to be essential for maximal milk production during lactation. Factors that interfere with these mechanisms can adversely affect mammary function during the impending lactation. A greater understanding of these processes may provide new approaches for increasing milk production in dairy cattle.     

Effects of oxytocin administration on oxytocin release and milk ejection

In experiment I, the effect of i.m. oxytocin (OT) injection (50 IU) on OT blood pattern was tested. Blood samples from 6 cows were collected for 2 h after OT injection either with or without milking. To test the effect of i.m. OT injection (50 IU) on milk ejection efficiency, intramammary pressure (IMP) was measured in 13 cows (experiment II). Milk ejection was induced by manual teat stimulation. After IMP increased and reached a plateau, OT was injected. In experiment III, the effect of chronic OT treatment on mammary gland sensitivity and endogenous OT release was tested. For 19 d, cows were i.m. injected at each milking with 50 IU OT (n = 13) or 5 mL of NaCl 0.9% (n = 14) 1 min before the start of udder preparation. To test mammary gland sensitivity, IMP recording was performed after a long (11 h, 7 OT cows, and 7 NaCl cows) and a short (3 h, 6 OT cows, and 7 NaCl cows) milking interval at d -1 and d 18. To test the effect of withdrawal of chronic i.m. treatment on OT release, blood sample collection was performed during evening milking at d 0 and d 19. Intramuscular oxytocin injection (experiment I) caused elevated OT blood levels observed at least for 2 h and showed an even more pronounced effect when milking was also performed. Intramuscular OT injection after teat stimulation (experiment II) caused additional milk ejection but only in 6 out of 13 cows. Withdrawal of chronic OT treatment (experiment III) did not reduce OT release during milking. However, ejection time was prolonged during OT infusion after a long milking interval. Ejection pressure tended to be lower after a short milking interval. It seems that the reduction of spontaneous milk removal after chronic OT treatment was due to reduced contractibility of myoepithelial cells in the mammary gland at a physiological range of OT concentrations.     

Pathological changes in bovine mammary glands following intramammary infusion of recombinant interleukin-2.

Eighteen lactating dairy cows were used to evaluate the physiological response of mammary glands to increasing doses of recombinant bovine interleukin-2. Right front and rear quarters were intramammarily infused with five different doses (.1 to 100 micrograms per quarter) of interleukin-2 as either a single or multiple treatment. Left front and rear quarters were intramammarily infused with a saline placebo and served as within-animal controls. Milk secretion samples for compositional analysis were collected from each quarter prior to infusion and at 12, 24, 36, and 48 h following infusion. Animals were slaughtered by exsanguination immediately following the 48-h sampling period, and mammary gland tissue was obtained for morphometric analysis. No changes in milk composition were observed between control quarters and those infused with up to 10 micrograms of interleukin-2 per quarter, administered as either a single or multiple treatment. Quarters infused with a single 100-micrograms dose of interleukin-2 or three consecutive doses of 25 and 100 micrograms of interleukin-2 had significantly lower lactose concentrations; there was a concomitant increase in bovine serum albumin, pH, and SCC compared with preinfusion concentrations or with control quarters. Morphometric analysis of tissue demonstrated an increase in stroma, a decrease in lumenal area, and a marked increase in the number of infiltrating leukocytes in those quarters infused with the higher doses of interleukin-2. Results suggest that interleukin-2 can be intrammammarily infused at doses as high as 10 micrograms per quarter without adversely affecting milk quality or normal mammary gland function.     

Determination of milk and mammary tissue concentrations of ceftiofur after intramammary and intramuscular therapy

Twenty-five Staphylococcus aureus strains isolated from bovine mastitis were tested for their susceptibility to ceftiofur. Zone diameter for 30 micrograms disks averaged 39 mm, and minimum inhibitory concentrations ranged from .5 to 1 microgram/ml. Tissue and milk concentrations were determined from biopsy and quarter milk samples collected from eight cows treated with either intramammary infusion of 100 or 200 mg of ceftiofur, one or two intramuscular injections of 500 mg of ceftiofur, or combination therapy of intramammary infusion coupled with intramuscular injection. Three additional cows received two intramammary infusions of 200 mg of cephapirin at 24-h intervals. Intramuscular injections of ceftiofur resulted in tissue and milk concentrations below detectable limits. Staphylococcus aureus was not eliminated from infected mammary glands by infusion of 100 mg of ceftiofur or by injection of 500 mg of ceftiofur by 48 h after treatment. Combination therapy of 100 mg of ceftiofur infused and 500 mg injected reduced S. aureus numbers in milk and tissue markedly, as did infusion of 200 mg of ceftiofur. Cows receiving intramammary infusion of 200 mg of ceftiofur (two doses at 24-h intervals) had highest concentrations in milk (450 micrograms/ml at 4 and 6 h) and in tissue (.08 microgram/mg at 30 h). These concentrations are similar to those obtained with two 200-mg doses of cephapirin at 24-h intervals. Histologic analysis of mammary parenchymal tissues showed that combination therapy resulted in higher percentages of alveolar luminal area and lower percentages of interalveolar stroma compared with infusion or injection alone. Histology of quarters receiving combination therapy was not different from that of quarters receiving cephapirin infusion alone.     

Inhibiting prolactin by cabergoline accelerates mammary gland remodeling during the early dry period in dairy cows

The inhibition of prolactin release using cabergoline, a dopamine agonist, is an effective strategy to accelerate the changes in mammary secretion composition after drying-off. The objective of this study was to determine how cabergoline may affect mammary tissue remodeling during early involution. Holstein dairy cows were treated with either a single i.m. administration of 5.6 mg of cabergoline (Velactis, Ceva Santé Animale, Libourne, France, n = 7) or placebo (n = 7) at the time of drying-off. Mammary biopsy samples were collected 1 wk before drying-off (d ?6), after 30 h of milk accumulation (d 1), and again 8 d following drying-off (d 8) to determine changes in gene expression, lactoferrin content, and cell turnover. Blood and mammary secretion samples were collected at d ?6 and again at d 1, 2, 3, 4, 8, and 14 following the abrupt cessation of lactation to evaluate indicators of blood-milk barrier integrity and other markers of mammary tissue remodeling. Cabergoline induced less SLC2A1, BAX, CAPN2, and IGFBP5 mRNA expression. In contrast, cabergoline did not modify changes in cell proliferation and apoptosis. Following the cessation of lactation, changes in mammary secretion composition (Na⁺ and K⁺) and blood lactose concentrations were indicative of a loss in the blood-milk barrier function in both treatment groups. Cabergoline treatment affected only Na⁺ and K⁺ concentrations at d 1, suggesting a moderate increase in tight junction permeability. The increase in the activity of MMP9 and in mammary epithelial cell concentration in mammary secretions was greater in cabergoline-treated cows than in control cows, suggesting more mammary tissue remodeling. The increase in lactoferrin immunostaining in the mammary tissue occurred earlier for cabergoline-treated cows than for control cows, and was essentially localized in the stroma. Changes in some key markers of mammary involution suggest that cabergoline accelerates mammary gland remodeling. Thus, a single injection of cabergoline after the last milking would facilitate drying-off by enhancing mammary gland involution.