|Acknowledgments: Paola Piantoni (Masters of Science, University of Illinois) contributed to the content of this page.|
Mammary Gland Involution
Involution is the process by which the mammary gland returns to its non-lactating state (Hurley, 1987). It could occur in a gradual fashion (after peak lactation or gradual decrease in suckling) or in an abrupt fashion (after cessation of milking or sudden weaning of the young) and it is regulated to a large extent by environmental factors (milking or suckling). Senile involution also has been described in humans (Lascelles and Lee, 1978).
Maintenance of Lactation Function
The lactation function of the mammary gland is maintained by a delicate balance of systemic blood-borne factors and local mammary-derived factors, many of which are directly affected by the process of milk removal. Systemic factors include galactopoietic hormones such as growth hormone and suckling-induced prolactin secretion which generally stimulate milk secretion, but also include factors arising from competing physiological states such as pregnancy which may inhibit lactation function. Additionally, local control of milk secretion is directly linked to physical removal of milk. The impact of these factors on mammary function is evident from the known effects of frequency of milk removal on milk yield, the effects of galactopoietic hormones on milk secretion, and the effects of milk stasis-induced mammary involution on mammary function. For a review of galactopoietic factors see the Galactopoiesis - Role of Hormones and the Galactopoiesis - Role of Milk Removal sections in the Lactation Lesson.
The role of galactopoietic hormones such as prolactin in maintenance of lactation is well established (reviewed by Tucker 1994), although specific cellular mechanisms of action continue to be investigated. Prolactin is considered the major galactopoietic hormone in nonruminants. Prolactin is released at the time of milk removal in ruminants and nonruminants, and it remains a key systemic modulator of milk secretion during lactation. Conversely, growth hormone is generally considered to be the predominant galactopoietic hormone in ruminants (Bauman 1992; Tucker 1994). Inhibition of prolactin secretion or administration of prolactin to lactating cows has little effect on milk yields (Karg and Schams 1974; Plaut et al. 1987). However, these apparently clear-cut roles of prolactin vs. growth hormone in maintenance of lactation in nonruminants vs. ruminants are probably an oversimplification (Wilde and Hurley 1996). For example, in lactating sheep both prolactin and growth hormone seem to be important for galactopoiesis (Hooley et al. 1978; Tucker 1994). Even in the rat, recent studies have demonstrated an important role for growth hormone, independent of the role of prolactin (Flint et al. 1992; Flint and Gardner 1994).
Regardless of the hormones involved, all attempts to evaluate milk secretion must account for continued removal of milk. This is a reminder of the critical role of local mammary factors in maintenance of milk secretion. One such factor that plays a major role in regulating milk secretion in many species is a feedback inhibitor of lactation (FIL) found in milk (Wilde et al. 1995). FIL is thought to be produced by the mammary cells as they synthesize and secrete milk. Accumulation of FIL in the milk-producing alveoli results in feedback inhibition of milk synthesis and secretion. Moreover, this short peptide was also shown to stimulate goat mammary cell apoptosis in vitro (Wilde et al., 1997; Wilde et al., 1999). Frequent removal of milk from the gland minimizes local inhibitory effects of FIL and increases milk secretion (Wilde et al. 1987; Wilde and Knight 1989; Wilde and Peaker 1990). Other proteins/peptides have been associated with this autocrine feedback mechanism that results in milk secretion being a self-limiting process. Even though much study has been done regarding this process, the specific trigger/s for mammary gland involution is/are still unknown.
Lactation in Decline
In spite of continued milk removal (with associated removal of FIL and stimulation of a post milking prolactin surge), milk yield in dairy cattle declines as lactation progresses. This decline occurs even with routine administration of growth hormone (bovine somatotropin). Lactation persistency is of particular concern in the production of milk. Mammary tissue function declines after peak lactation and this is at least in part due to a decrease in mammary cell number (Knight and Peaker 1984; Wilde and Knight 1989). The cell loss during the declining phase of lactation in the goat and cow apparently is the result of programmed cell death, also called apoptosis (Quarrie et al., 1994; Wilde et al., 1997). The mechanisms that control lactation decline remain important areas of investigation. Mammary involution is a greatly enhanced extension of these processes leading to a complete cessation of lactation function.
Concurrent pregnancy also influences persistency of milk yield in the declining phase of lactation. Inhibitory effects of pregnancy on lactating cows do not become apparent until about mid pregnancy (Wilcox et al. 1959; Bachman et al. 1988). An inhibitory effect of pregnancy on lactation has been noted in a number of other species, as well (Wilde and Knight 1989; Tucker 1994). The mechanism of this effect is not fully understood. However, the timing of inhibition of milk yield in cattle coincides approximately with the period of increasing placentally-derived plasma estrogen (Robertson and King 1979). Estrogen may have an effect on the transition of mammary function from a lactating state to an involuting state (Athie et al. 1996; Bachman 1982).
Bovine Mammary Gland Involution
Cessation of milk removal leads to rapid changes in the mammary tissue and initiation of the process of mammary involution (Hurley 1989). Changes in composition of mammary secretions during the early phases of involution indicate rapid changes in the normal mechanisms involved in milk synthesis and secretion (see discussion below; also Hurley and Rejman 1986; Hurley 1987; Hurley et al. 1987; Rejman et al. 1989; Hurley 1989). These changes in mammary gland secretion composition include a rapid decline in lactose concentration in the mammary secretions, indicating that lactose synthesis, and the associated water transport mechanism, decline soon after cessation of milk removal. However, total protein concentrations increase in early involution, partially because of water resorption from the secretion and partly due to increased concentrations of lactoferrin, serum albumin and immunoglobulins. Lactoferrin is a major protein found in mammary secretions during involution (Rejman et al. 1989). Its synthesis is increased during involution in contrast to milk-specific proteins such as casein whose synthesis is decreased (Hurley and Rejman 1993; Hurley et al. 1994a). Lactoferrin has a number of potential functions in the mammary gland, particularly as a nonspecific disease resistance factor (reviewed by Sanchez et al. 1992).
Involution-associated ultrastructural changes in bovine mammary cells begin within 48 hours after cessation of milk removal (Holst et al. 1987; Hurley 1989). The most apparent change is the formation of large stasis vacuoles in the epithelial cells (Holst et al. 1987), formed largely as a result of intracellular accumulation of milk fat droplets and secretory vesicles (Hurley 1989). These vacuoles persist to at least 14 days of involution and are usually gone by day 28 (Holst et al. 1987). Alveolar lumenal area declines during this period, while interalveolar stromal area increases. A substantial reduction in fluid volume in the gland occurs between day 3 and 7 of involution (Hurley 1989), probably accounting for the reduction in lumenal volume. By day 28 the collapsed alveolar structures remaining are considerably smaller than during lactation, with a very small lumen. General alveolar structure is maintained thoughout involution in the cow.
Histological and ultrastructural work on the bovine mammary gland during involution (Holst et al. 1987; Hurley 1989) provides no evidence for the extensive tissue degeneration observed in other species, such as rodents and others (Helminen and Ericsson 1968a,b; Helminen and Ericsson 1971). In cattle, changes in mammary gland micro-structure are more a reflection of a change in secretory state than a change in tissue regression. Some researchers associated this to the fact that this species also is typically pregnant during involution and pregnancy might play a role in the inhibition of epithelial cell apoptosis, as observed in rodents (Capuco and Akers, 1999). Several previous studies involving mammary tissue to study involution in cows involved pregnant and non-pregnant animals (Holst et al., 1987; Akers et al., 1990). Thus, it remains to be clarified whether pregnancy itself affects histological differences between rodents and cows during mammary gland involution and in which extent.
In cows, limited autophagocytic processes occur only transiently during the initial two days after cessation of milking. On the other hand, formation of autophagocytic structures in rodent mammary tissue is characteristic of involution (Helminen and Ericsson 1968a,b; Helminen and Ericsson 1971). Moreover, a detachment of epithelial cells from the basement membrane and their loss from the tissue has been reported in rodents and other species (Wellings and DeOme 1963; Verley and Hollman 1967; Helminen and Ericsson 1968b; Richards and Benson 1971). This leaves characteristic bare spaces on the basement membrane and myoepithelial cells are thought to fill the space. No such situations are observed in the involuting bovine gland (Holst 1987; Holst et al. 1987). More recently, the involution process in the mouse has been characterized by examining the role of apoptosis.
In summary, histological changes occurring in the involuting bovine mammary gland involve:
Apoptosis and Mammary Gland Involution
Mammary involution in the mouse is characterized by a rapid loss of tissue function and degeneration of the alveolar structure and massive loss of epithelial cells. This cell loss has been related to programmed cell death I (PCD I)or apoptosis (cell shrinkage and margination and condensation of chromatine) (Strange et al. 1992, Walker et al. 1989). Another type of programmed cell death that has been implicated in mammary involution in cows is PCD II or autophagy (autophagosomes and autophagic vacuoles) (Gajewska et al. 2005). Apoptosis, which is usually considered to be the prevalent form of PCD controlling viability and homeostasis during development, is both a natural and systematic method of cell suicide which takes place during normal morphogenesis, tissue remodeling, and in response to infection or irreparable cell damage (Wyllie et al. 1984, Schwartzman & Cidlowski, 1993).
There are two distinct types of cell death, the above mentioned programmed cell death (apoptosis and autophagy) and the accidental cell death (necrosis), which may be distinguished by morphological, biochemical, and molecular changes in dying cells. The process of apoptosis was originally distinguished from necrosis on the basis of its ultrastructure (Kerr 1971, Kerr et al. 1972). Apoptosis may be identified by a characteristic pattern of morphological changes: nuclear and cytoplasmic condensation, nuclear fragmentation, and formation of apoptotic bodies (Walker et al. 1989, Strange et al. 1992). These changes are associated with cleavage of chromatin into discrete sized oligonucleosome fragments by a calcium dependent endonuclease (Arends et al. 1990), resulting in the appearance of oligonucleosomal DNA laddering in ethidium bromide stained gels (Wyllie et al. 1980).
Morphology consistent with apoptotic cell death can be observed in the murine mammary gland within two days of milk stasis. The nucleus and cytoplasm condense, the chromatin becomes fragmented and marginated, and apoptotic bodies are formed (Walker et al., 1989; Strange et al., 1992). This cell loss results in extensive disintegration of alveolar structure during the early period of involution in the mouse. DNA laddering characteristic of apoptosis also has been detected in goat mammary tissue during early and late lactation (Quarrie et al., 1994) and during late lactation in the cow (Wilde et al., 1997). This would suggest that removal of secretory epithelial cells by apoptosis is a normal physiological event in the ruminant mammary gland, even during lactation. In addition, milk stasis has been demonstrated to stimulate DNA laddering in both goat and cow mammary tissue (Quarrie et al., 1994; Wilde et al., 1997). These observations suggest that mammary epithelial cells are indeed lost during involution in the bovine mammary gland. However, this process of cell loss does not seem to be as dramatic as that observed in the mouse. In spite of the loss of cells, bovine mammary alveoli retain general structural integrity throughout involution (Holst et al. 1987). While the role of cell loss in the mouse mammary gland during involution is clear, the impact of mammary apoptosis in the bovine is not fully characterized.
In cows, during active involution there is an increase in the ratio of epithelial cell death to epithelial cell proliferation, while during the preparation of the gland for a new lactation there is a decrease in the ratio of epithelial cell death to epithelial cell proliferation (Sorensen et al., 2006). This dry period in the cow is characterized more by cell turnover (increased apoptosis and proliferation, which could be related to the concurrent pregnancy) than by programmed epithelial cell death and remodeling, as observed in the mouse (Capuco and Akers, 1999).
The remaining discussion deals primarily with the dry period of the dairy cow. Involution marks only the initial phase of the dry period.
Dry Period and Subsequent Lactation
*** The mammary gland of the dairy cow requires a nonlactating (dry) period prior to an impending parturition to optimize milk production in the subsequent lactation: the mammary gland needs to recover from the previous lactation and prepare for the next by repairing or replacing senescent or damaged epithelial cells.
The optimum length of the dry period has been further evaluated to consider number of lactations. Regarding the required time for this process to occur, a period of 41 to 45 d between 1st and 2nd lactation is recommended. This length of time will maximize milk yield across these two lactations, although there does not seem to be an associated loss with dry periods >45 d. On the other hand, it has been suggested that a period of at least 55 d will be needed between 2nd and 3rd and 3rd and 4th lactations to maximize milk yield across the 3rd and 4th lactations. When lifetime yield is considered, a dry period between 30 and 60 d is recommended (Kuhn et al. 2006).
The normal procedure to dry off a cow is to withdraw all grain (give diets higher in fiber) and reduce the water supply several days before the start of the dry period. Reduction of the water supply during dry-off is still very controversial. These procedures drastically reduce milk production during that time. Another method would be to dry-off abruptly and feed only hay and water for a few days. After dry-off, milking is halted about 45 to 50 d before expected date of parturition.
At this time, dry-off therapy (antibiotic treatment) is widely used. The dry-off therapy consists of an udder infusion with slow-release antibiotics (effective levels of antibiotics remain in the udder for 21 d or more) which can help prevent infections that may occur in early involution and/or treat existing subclinical infections of the udder produced by microorganisms such as Staphylococcus bacteria. After antibiotic treatment, teat sealants could be also used to prevent new intramammary infections. Teat sealants provide an extra physical barrier to the udder, preventing bacteria from entering the teat canal and therefore, reducing the incidence of clinical mastitis for the entire dry period. It is noteworthy that cows with clinical mastitis should NOT be dried off until mastitis is resolved.
After milking is stopped intramammary pressure increases, milk products accumulate in the gland and further milk secretion is inhibited. Sometimes, if the udder becomes extremely congested, it may need to be re-milked. However, this practice stimulates further milk synthesis because intramammary pressure is reduced and pituitary hormones (oxytocin and prolactin) are released. Perhaps more importantly re-milking removes the leukocytes from the udder at a time when many are needed to prevent infection. Nevertheless, if signs of clinical mastitis are seen after dry-off, the cow should be milked out and treated a second time after a week. It usually is unnecessary to re-milk if production is reduced below about 50 lbs per day before milking is stopped.
In studies with identical twins, milked 2X/day the twin with no dry period gave only 62-75% as much milk as the twin with 60 day dry period.
If you milk 1/2 of the udder throughout the dry period while the other 1/2 is dry, then the milked side gives less milk in the subsequent lactation. (DNA concentrations are the same in both halves)
Dry Period and Mastitis
The early dry period ( first week or two) is the time of the highest incidence of new intramammary infection (new IMI). The mid-dry period is the time of lowest incidence of new IMI. The peripartum period is also a time of high incidence of IMI. For a review see the Mastitis Lesson.
Mammary Physiology During the Dry Period
Physiology of the mammary gland during the dry period differs markedly from that during lactation. For the sake of convenience, the dry period can be divided into three phases :
Active involution begins with the cessation of periodic milk removal, either by drying off a cow or by weaning the young. Halting milk removal results in milk stasis. Halting milk removal results in milk stasis and udder distension and both factors have been postulated as probable causes of mammary involution through local chemical feedback by milk constituents, formation of other substances in stored milk, mechanical stress and stretching of the cells that lead to the loss of secretory activity (Peaker, 1980; Hurley, 1989; Li et al., 1999). In the cow active involution is probably complete by 21 to 30 days after dry off. It is a transition phase of the mammary gland from the lactating to the nonlactating state. Changes in the mammary secretion volume and composition occur during active involution, including:
Milk continues to accumulate for a couple of days after drying off. Volume of secretion in the gland increases for 2 to 4 days after drying-off, then declines rapidly over the next week. Fluid volume continues to decrease through ~30 days (in a 45-60 day dry period).
Concentrations of milk-specific components (caseins, a-lactalbumin, ß-lactoglobulin, and milk fat) decline slowly during the first 2-3 wk of the dry period, but never completely disappear. Lactose concentrations decline rapidly (Aslam et al. 1994; Hurley and Rejman 1986; Hurley 1987; Hurley et al. 1987).
Proteins of serum origin (immunoglobulins, serum albumin) increase in concentration during the first week of involution. All classes of immunoglobulins (Ig) increase, including IgG1, IgG2, IgA, IgM. There is a transitory increase in the selective transport of IgG1 from about days 2 to 4 of involution, but not as much as during colostrum formation. Increased serum albumin (SA) concentrations reflect increased permeability (tight junctions between epithelial cells are loosened for a period of several days). Albumin concnetration does not approach serum levels or levels found in the milk during acute inflammation, therefore, the permeability barriers are not completely destroyed.
NAGase (N-acetyl-ß-D-glucosaminidase) activity increases substantially during mammary involution. NAGase is an intracellular lysosomal enzyme that is secreted mainly by polymorphonuclear leukocytes, such as neutrophils, during phagocytosis and, to some degree, by damaged epithelial cells during cell lysis. This indigenous enzyme is found in large quantities in the mammary gland during involution and inflammation. The specific function of NAGase in the gland is not known, but its activity in mammary secretions sometimes is used as an indicator of tissue changes that accompany involution and inflammation, so it is being currently considered as a parameter to monitor subclinical mastitis (Pyorala 2003).
Lactoferrin (LF) concentrations increase markedly during active involution (Rejman et al. 1989). LF is an iron-binding protein, thought to compete with bacteria for iron (see Sanchez et al. 1992). This is the basis for lactoferrin's bacteriostatic action, although recent evidence suggests that other properties of LF may account for its bacteriostatic action. Invading bacteria are forced to compete with LF for iron. For coliform bacteria it is the citrate:LF ratio that is important. Citrate chelates Fe and bacteria can use the citrate-iron complex. During early involution citrate concentration declines, while LF concentration increases. LF may play a role in mammary gland by affecting phagocyte function. LF also may limit the oxidative degeneration of cellular components that can occur during periods of tissue disruption such as during inflammation and involution.
Cells in secretions of the involuting mammary gland:
Very few cells (less than 2%) in mammary secretions during involution are epithelial cells. Most cells in the secretion are leukocytes. Total leukocyte concentrations in mammary secretions increase rapidly in early involution.
Types of leukocytes found in mammary secretions during the dry period:
PMN (polymorphonuclear neutrophils). These are phagocytic leukocytes. They predominate for the first 3-7 days (if the quarter is also infected, then they will predominate throughout the early stages of infection).
Macrophages are the predominant cell type after ~7 days. These are also phagocytes. Many are filled with ingested fat droplets and other debris. They play a major role in removing large quantities of fat and cellular debris, including dead PMN. Macrophages are also the predominant cell type in the colostrum at parturition.
Lymphocytes are always present. They increase in proportion roughly in parallel with the macrophages, but may only become the predominant cell type during the mid-dry period. Specific function of lymphocytes in the involuting mammary gland is unknown.
New Intramammary Infections During Active Involution
A number of factors contribute to the elevated susceptibility to infection during the early dry period:
Streak Canal and Udder Fluid:
Phagocytic Leukocytes (Somatic Cells) and Other Defenses:
Intramammary dry-cow antibiotic therapy, given immediately after the last milking, is very effective in controlling mastitis in early involution (except for coliform mastitis caused by IMI). Dry-cow antibiotic therapy is the most effective preventative treatment to help the udder get through the early stages of involution.
Steady State Involution
The length of the steady state period depends on the total length of the dry period. If active involution takes about 4 weeks to complete in the dairy cow and the redevelopment stage takes about 3 or 4 wk. These periods will then account for the recommended optimal 45-60 day dry period. So, cows with a 45-60 day dry period probably have a very short steady state phase or no steady state phase of involution at all. When the dry period is less than 40 days, the tissue is undergoing active involution and beginning the redevelopment phase concurrently. This may contribute to the decline in optimal milk yield in the next lactation. However, other factors (metabolic and management factors) also contribute to the requirement for the 45-60 day dry period.
New IMI are generally low during the steady state phase period. This is the period of greatest resistance to intramammary infection. If an infection occurs, it usually is spontaneously eliminated.
Streak Canal and Udder Fluid:
Phagocytic Leukocytes (Somatic Cells) and Other Defenses:
Redevelopment and Colostrogenesis
Prepartum Period : Colostrogenesis and Lactogenesis
This phase of the dry period marks the transition from the nonlactating state to the lactating state. We do not know exactly when this period begins, but it is probably beginning at about 3 to 4 weeks prepartum. Recent work indicates that in cows given a 60 day dry period, an increase in mammary DNA synthesis begins occurring about 35 days prepartum (Capuco et al. 1997). This might indicate that the early stages of redevelopment are beginning even 5 weeks prepartum.
The selective transport of IgG1 is a major activity of the epithelial cells during the last 2 wk prior to parturition (see the immunoglobulin transport section in the Mother & NeonateLesson). Concentrations of the major milk components increase beginning ~2 wk prepartum, and then increase markedly ~3-5 days prepartum. See Lactogenesis in the Mother & Neonate Lesson for more on the two stages of lactogenesis.
The potential for IMI is increased again because the gland is undergoing the opposite changes from those during early involution.
Streak Canal and Udder Fluid:
Phagocytic Leukocytes (Somatic Cells) and Other Defenses:
In summary, citrate increases and LF concentrations are relatively low, leukocyte numbers are fairly low, phagocytic capacity of phagocytes is reduced again because of increased numbers of fat droplets, so the incidence of new IMI is again high. (Dry-cow therapy does not last this long).
Summary of Changes in Composition of Mammary Secretions During the Dry Period
Mammary Development Resources