Mammary Involution Research Articles

Mammary Gland Function During Involution

The process of mammary gland involution occurs during the transition from a lactating to a nonlactating state. This transition phase begins after cessation of milk removal and results in changes in mammary secretion composition. Secretion volume declines during involution, as does the concentration of most milk-specific components. Lactoferrin, hydrolytic enzymes, immunoglobulins, and serum-derived components increase in concentration in the secretions during involution. Changes in mammary secretion composition may reflect changes in function of alveolar epithelial cells and have implications for the disease resistance of the gland. Histological and ultra-structural changes occurring in the gland are consistent with a decline in secretion of milk components from epithelial cells. Autophagocytic mechanisms may be involved in this decline in the lactation function. Ultrastructurally, there is little evidence for an extensive loss of epithelia in the bovine mammary gland during involution. Completion of the functional changes occurring in the gland during the process of involution may be required for the gland to redevelop fully for maximal milk yield in the subsequent lactation. Cellular mechanisms involved in mammary involution and relationships between the processes of involution and redevelopment should be areas of particular interest in the mammary function of dairy cattle.

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.

Immunological Aspects of Mammary Involution

Frequency of new intramammary infection is greatest during early involution, decreases during middle stages, and then increases prepartum. Penetrability of the teat canal, antibacterial properties of keratin, bacterial adherence, and epithelial sensitivity to toxins play a role in resistance. Leukocytes phagocytose bacteria and regulate expression of immune mechanisms, although their function is compromised during certain stages of involution. These cells increase to millions per milliliter as involution progresses and then decrease prepartum. Macrophages predominate in lacteal secretions, followed by lymphocytes and neutrophils. Lactoferrin, a major whey protein and iron chelator, is also associated with resistance to infection during the nonlactating period and may have immunomodulatory properties. Lacteal immunoglobulins increase throughout involution peaking prepartum and function by opsonizing bacteria, neutralizing toxins, and preventing bacterial adherence. Immunoglobulins are derived from blood or are produced locally by plasma cells present in the subepithelial mammary stroma. Plasma cells, lymphoid cells, and other protective leukocytes present in teat end tissues accumulate during infection and concentrations increase in response to local antigenic stimulation. Various aspects of the mammary immune system are compromised during periods of functional transition. Thus, vaccination, immunostimulation, accelerated involution, and intramammary devices are some methods now being tested to amplify local immunity and protect the gland from bacterial infection.

Enzyme-Linked Immunosorbent Assays of Bovine Lactoferrin and a 39-Kilodalton Protein Found in Mammary Secretions During Involution

Enzyme-linked immunosorbent assays were developed for two proteins found in mammary secretions during the non-lactating period: lactoferrin and a 39-kdal protein. Minimum detectable concentrations were .12ng/ml for lactoferrin and .0037ng/ml for the 39-kdal protein. Standard curves were linear in the ranges of .4 to 100ng/ml for lactoferrin and 2.4 to 30ng/ml for the 39-kdal protein. The immunoassays were used to measure concentrations of the proteins in mammary secretions during involution. From the last day of milking to d 30 of involution, mean concentrations of lactoferrin and the 39-kdal protein increased from .82±.35 to 78.5±12.43 mg/ml and from 11.8±3.8 to 84.4±21.1µg/ml, respectively. The immunoassays described will be valuable for further studies on the synthesis and secretion of these proteins during mammary involution.

Evaluation of an anti-inflammatory factor derived from hyperimmunized cows.

An anti-inflammatory factor isolated from milk of hyperimmunized cows was analyzed in vitro and in vivo. Macrophages collected from lacteal secretions of a unimmunized nonlactating cow showed increased ability to kill phagocytosed Staphylococcus aureus when incubated with the anti-inflammatory factor. Mice injected intraperitoneally with 10 mg/kg of anti-inflammatory factor demonstrated an increased LD50 to S. aureus when challenged intraperitoneally. Injected mice also demonstrated significantly (P less than 0.05) less mammary inflammation and involution and increased clearance of S. aureus when challenged intramammarily. Quantitative histologic analysis of mammary tissues from mice injected with anti-inflammatory factor demonstrated significantly (P less than 0.05) more lumen, less interalveolar connective tissue, and less leukocytic infiltration compared with control mice. Mammary glands of mice injected with anti-inflammatory factor and challenged with S. aureus also contained fewer colony-forming units than control mice. The product appeared to exert its effect on the nonspecific defense system via modulation of leukocyte function.

Studies on the inflammatory response during involution of the ovine mammary gland.

Cessation of lactation, initiated by the removal of lambs from their mothers, induced an inflammatory response with elevated numbers of neutrophil leucocytes in mammary secretions being detected within 16 h. Leucocyte numbers were still elevated 7 d later. Infusion of whole milk into non-lactating glands caused no change in leucocyte numbers after 24 h. Involution secretions, however, had the capacity to induce an inflammatory response when infused into non-lactating glands. Daily infusion of endotoxin (100 ng) into non-lactating glands resulted in a marked attenuation of the inflammatory response. Such desensitization did not accompany reinfusion of involution secretion. Responses to involution secretion were enhanced in glands desensitized to endotoxin. The failure of desensitization to develop in glands receiving repeated infusions of involution secretion and the lack of cross-desensitization between involution secretion and endotoxin indicate that a novel mediator of inflammation may be produced during mammary involution in sheep.

Electrolytes and free fatty acids in milk during mammary involution in the cow

Measurements have been made of the concentrations of triglycerides, free fatty acids, sodium, chloride and potassium in milk samples obtained from cows during acute mammary involution and after parturition. Free fatty acid levels in milk increased more than 10-fold during involution. Their appearance did not immediately follow the cessation of milking but followed the increase in permeability of the mammary epithelium, evidenced by changes in the electrolyte content of the milk, that occurs after a delay of a few days. However, the concentration of free fatty acids did not remain high throughout the dry period but had declined to low levels before the change in permeability was reversed at the next parturition. It is concluded that the high level of free fatty acids in milk during mammary involution does not result from equilibration with an extra-mammary pool, but most likely arises from breakdown of triglycerides remaining in the gland which may be accelerated in some manner by the increase of permeability of the mammary epithelium.

Immunohistological localization of IgG1, IgA and secretory component in the bovine mammary gland during involution.

Immunoperoxidase methods were used to localize secretory component, immunoglobulin A and immunoglobulin G1 in mammary tissue from dairy cows. In lactating tissue, immunostaining for immunoglobulin A and secretory component was observed primarily in the luminal contents of alveoli. By day 2 of involution, alveolar epithelial cells stained for both immunoglobulin A and secretory component. Staining of alveolar epithelial cells for immunoglobulin A and secretory component continued throughout the period of mammary involution. No staining for secretory component was observed in the interalveolar stromal area. Immunoglobulin G1 immunostaining was localized primarily in the interalveolar areas in lactating tissue, but was localized at the apical and basolateral surface of alveolar cells on day 2 of involution. In contrast to immunoglobulin A, immunoglobulin G1 staining of epithelial cells did not persist and was primarily in the interalveolar areas by day 4. These results suggest that an increased localization of immunoglobulin G1 in bovine mammary epithelial cells may occur transiently in early involution, while an increase in immunoglobulin A and secretory component localization in epithelial cells persists throughout involution.

Origin, fate, and properties of multinucleated giant cells and their association with milk-synthesizing tissues of the bovine mammary gland

Histological study of multinucleated giant cells (MGC) in bovine mammary glands demonstrated that the cells predominated during mammary involution. Concentrations of MGC increased from the early to later stages of involution, and were more prevalent in older cows. There was no association between infection status and prevalence of MGC. A study of the relationship between MGC and other components of the mammary parenchyma indicated that these cells formed in response to substances that accumulated in mammary secretions during involution. MGC appeared to play a role in resorption of insoluble milk constituents, and removal of amyloid and effete macrophages.

Secretion composition during bovine mammary involution and the relationship with mastitis

1. 1. Bacteriological analysis revealed that 30% of quarters contained coagulase-negative staphylococci, Staphylococcus aureus, Corynebacterium bovis, or streptococci. 2. 2. As involution progressed, somatic cell counts, percent protein, pH, and concentrations of serum albumin, lactoferrin, and immunoglobulin G increased while percent fat, concentrations of citrate, and the citrate to lactoferrin molar ratio decreased. 3. 3. Mammary secretion from infected quarters had significantly higher numbers of somatic cells, percent polymorphonuclear leukocytes, and pH, but lower percentage lymphocytes, fat, and lactoferrin concentrations compared to uninfected quarters. 4. 4. Results suggest intramammary infection altered normal secretion composition during bovine involution and lactogenesis. 5. 5. Lower levels of antibacterial components in bovine mammary secretion during the peripartum period may have reduced the natural defense potential of the gland.

Phosphoribosyl pyrophosphate and phosphoribosyl pyrophosphate synthetase in rat mammary gland. Changes in the lactation cycle and effects of diabetes, insulin and phenazine methosulphate

Changes in the tissue content of phosphoribosyl pyrophosphate (PPRibP), glucose 6-phosphate, ribose 5-phosphate (Rib5P), RNA and DNA, of the activity of PPRibP synthetase (EC 2.7.6.1) and the conversion of [1-14C]- and [6-14C]-glucose into 14CO2 were measured at mid-lactation in the normal and diabetic rat and in pregnancy, lactation and mammary involution in the normal rat. The PPRibP, glucose 6-phosphate and Rib5P contents increase during pregnancy and early lactation to reach a plateau value at mid-lactation, before falling sharply during weaning. The PPRibP content, PPRibP synthetase activity and flux of glucose through the oxidative pentose phosphate pathway (PPP) all change in parallel during the lactation cycle. Similarly, after 3 and 5 days duration of streptozotocin-induced diabetes, ending on day 10 of lactation, there were parallel declines in PPRibP content, PPRibP synthetase and PPP activity. The effect of streptozotocin was prevented by pretreatment with nicotinamide and partially reversed by insulin administration. Addition of insulin to lactating rat mammary-gland slices incubated in vitro significantly raised the PPRibP content (+47%) and the activity of the PPP (+40%); phenazine methosulphate, which gives a 2-fold increase in PPP activity, raised the PPRibP content of lactating mammary gland slices by approx. 3-fold. It is concluded that Rib5P, generated in the oxidative segment of the PPP, is an important determinant of PPRibP synthesis in the lactating rat mammary gland and that insulin plays a central role in the regulation of the bioavailability of this precursor of nucleotide and nucleic acid synthesis.

Morphological changes caused by experimental Streptococcus uberis mastitis in mice following intramammary infusion of pokeweed mitogen.

Mice were used as models for bovine mastitis in an attempt to modify the susceptibility of mammary glands to Streptococcus uberis infection. Murine mammary glands were injected with pokeweed mitogen (PWM) prior to experimental bacterial challenge to accelerate involution and enhance antimicrobial mechanisms. PWM injection reduced the numbers of streptococci recovered when compared to controls. Histological examination of tissues from PWM-treated mice revealed a reduction in secretory activity and advanced involution. PWM-treated tissues had considerably more leukocytes infiltrating the epithelium, lumen, and underlying connective tissue. Bacteria were observed within the epithelium and alveolar lumen and internalized within neutrophils and macrophages in both PWM-injected and control tissue. Results of this study suggest PWM injection provided some protection against S. uberis mastitis by accelerating mammary involution, enhancing antimicrobial defenses, and facilitating a marked cellular response prior to bacterial challenge.

A study in vivo of mammary differentiation and involution during a normal lactation cycle in the mouse

A study <i>in vivo</i> of mammary differentiation and involution during a normal lactation cycle in the mouse

Role of macrophages and multinucleate giant cells in the resorption of corpora amylacea in the involuting bovine mammary gland.

Microscopic examination of involuting bovine mammary tissue revealed elevated concentrations of corpora amylacea in alveolar lumina. Morphologic relationships between amyloid bodies, macrophages, and multinucleate giant cells (MGCs) suggested phagocytosis and degradation of the deposits by the phagocytic cells. Resorption of amyloid material by macrophages and MGCs during the process of mammary involution may be instrumental in preventing accumulation of corpora amylacea in secretory tissue which may interfere with mechanisms of milk synthesis and secretion.

Biochemical and Antibacterial Properties of Bovine Mammary Secretion During Mammary Involution and at Parturition

Mammary secretions from four Holstein Friesian cows were collected during late lactation, early involution, and early in subsequent lactation. Changes of pH, concentration of serum albumin, immunoglobulin G, citrate, lactoferrin, and number of leukocytes in secretions were typical of milk from glands undergoing these physiological transitions. Whey prepared from a cow's secretions was evaluated for its capacity to inhibit growth of Escherichia coli, 60-Lilly, and a coliform strain isolated from mammary secretions of that cow. Wheys from different glands of the same cow differed markedly in their capacity to inhibit growth of coliforms. Inhibition of both strains by the wheys increased significantly during the dry period and was maximal in wheys collected day 15 of the dry period and at parturition in the subsequent lactation. The coliform strain isolated from a specific cow was inhibited more than Escherichia coli, 60-Lilly, by whey from the specific cow. Inhibition of the cow-specific coliform strain by the day 15 whey was reduced significantly after addition of ferric iron or sodium citrate. Addition of excess ferric iron or citrate to wheys collected at parturition did not alter significantly their inhibitory capacity within cow. However, reduction of growth inhibition was significant when data from the four cows were pooled.

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.

Half-life, clearance and production rate for oxytocin in cattle during lactation and mammary involution I

Basal concentration, production rate, metabolic clearance rate and T 1 2 for oxytocin were measured in Holstein cows at three lactational stages and during mammary involution. At each lactational stage, oxytocin was given intravenously at .5 IU/min or 1.0 IU/min for 60 min. Infusions were preceded by priming. During involution, the high dose was used. Mean basal concentrations of oxytocin ranged from 8.7 to 21.4 uU/ml. Mean basal values at early, middle, late lactation and involution were 17.57, 12.33, 15.15, 21.13 uU/ml, respectively and differed significantly. The mean “rapid” T 1 2 was 3.87 ± .1 min. Early, middle, late lactation and involution T1 2 s were 4.2, 3.7, 4.0 and 3.5 min, respectively. The rapid T 1 2 was not affected by lactational stage. The mean “slow” T 1 2 was 25.53 ± 1 min. Early lactation and dry period means differed significantly. The overall mean oxytocin clearance rate was 8.41 ± .1 ml/kg ·min. Clearance rate declined through lactation and into the dry period. Mean values of 9.3, 8.8, 7.8 and 7.1 ml/kg ·min were obtained at early, middle, late lactation and involution, respectively. Clearance rates at late lactation and involution differed significantly from one another and from the early and middle stages. Mean entry rates for oxytocin at early, middle, late lactation and involution were 168.79, 106.03, 111.1 and 146.8 uU/kg ·min, respectively. Measurements at early lactation and involution were greater than values for middle and late lactation. To summarize, basal oxytocin concentrations can be measured in cows that are lactating or undergoing mammary involution and changes in concentrations during lactation are related to hormone production (entry rate) and metabolic clearance rates.

Physiological and pharmacological studies of oxytocin in virgin cattle III

Resting concentrations of oxytocin were measured in virgin cattle and it was determined if these animals would release oxytocin in response to milking associated stimuli. Half-life, clearance and entry rates for oxytocin were also quantified, to compare previous data from experiments carried out on lactating and nonlactating cows. Six Holstein virgin heifers received a two minute udder massage. Blood samples were taken before, at and after massage and analyzed for oxytocin by radioimmunoassay. Resting concentrations of oxytocin averaged 24.8 ±2.27 uU/ml (mean ± SE) and increased to 28 ±2.3 uU/ml at 2 min. The increase in oxytocin was clearly apparent after post-stimulation concentrations were corrected for basal (pre massage) levels. Serum oxytocin concentrations remained below baseline for 25 min post stimulation. No increase in oxytocin was seen prior to udder stimulation. Heifers were infused with 1.0 IU/min oxytocin to measure pharmacokinetic parameters. Oxytocin disappearance from sera was monitored by radioimmunoassay. A two compartment model for oxytocin elimination produced two half-life components. A rapid half-life component with a mean value of 3.07 ±.32 min and a slow half-life component with a mean of 24.3 ±2.2 min. The overall clearance rate for oxytocin was 9.4 ±.58 ml/kg min. The mean entry rate of oxytocin into the body was 231.96 ±25.96 uU/kg min. The sensitivity of the neuroendocrine reflex, eliciting oxytocin release, appears dependent upon the physiological state of the animal. Half-life and clearance measurements for oxytocin compare closely to those obtained in previous studies for cows in either early lactation, late lactation or undergoing mammary involution. It appears that the clearance and production of oxytocin are also related to the physiological state of the animal.

Selective transport of serum-derived IgA into mucosal secretions.

The relative contribution of serum-derived and locally produced IgA has been estimated in sheep mammary secretion at various stages of lactation by comparing the transport of radiolabeled IgA and IgG2 from serum to secretions. During early and mid-lactation, but not during involution, serum IgA was selectively transported to mammary secretion on a large scale; in comparison, there was no transport of serum IgA into intestinal secretion. Estimates of local production indicated that the bulk of the IgA in mammary involution secretion and intestinal secretion was locally produced, whereas there was no evidence for local production of IgA in early or mid-lactation secretion. Our studies reveal an inverse relationship between the extent of local production of IgA by plasma cells underlying secretory epithelia and the selective transport of IgA from serum. This finding suggests that selective transport of serum IgA occurs at a number of mucosal sites, but is dependent on secretory component availability, because where local production is predominant, there are fewer secretory component molecules available for serum IgA transport. For this reason, bile transport of IgA does not represent a unique mechanism for the removal of IgA from serum.

Bovine Mammary Involution Following Intramammary Infusion of Colchicine and Endotoxin at Drying Off

Effects of intramammary infusion of colchicine, endotoxin, and colchicine in combination with endotoxin on mammary involution were studied on 25 dairy cows in late lactation. Both quarters of right udder halves were infused at or near drying off while left udder halves received no infusion. Samples of mammary secretion were obtained 1 day prior to drying off, at drying off, and .5, 1, 2, 3, and 7 days post drying off. Mammary secretion obtained during the early dry period from udder halves infused with colchicine or colchicine and endotoxin at or near drying off had elevated somatic cells, lactoferrin, serum albumin, immunoglobulin G, and pH as compared to left untreated udder halves. Citrate, molar ratio of citrate to lactoferrin, and the index of selective accumulation of immunoglobulin G were lower in infused than uninfused udder halves. Fluid volume removed from udders at 7 days involution was 40% less in udder halves infused with colchicine or colchicine and endotoxin as compared to uninfused udder halves. Intramammary infusion of colchicine and endotoxin at drying off accelerates bovine mammary involution.