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14. LACTATION

EMBRYONIC AND FETAL DEVELOPMENT OF THE MAMMARY GLAND During embryonic development, the glandular tissue, which will form the mammary gland mesenchyme, is derived from the ectoderm. In the bovine, by day 30 of gestation, ectodermal cells condense and line up at both sides of the abdomen between the limbs and by day 35 this becomes the mammary line (Fig. 14-1).

Subsequently, these lines shorten and, grow more attached and deeper into the mesenchymal cells, forming a crest. At 43 days of age, this structure becomes

lenticular and then spherical; by now they are called mammary buds (Fig. 14-2).

The crown rump length of the conceptus is about 2.5 cm at this time. The spherical bud still grows deeper into the mesenchymal cells and starts becoming different for males and females. In the female, the spherical bud evolves into a conical structure (Fig.14-3), which by day 65 starts forming the teats.

By day 80, when the crown rump length is 12 cm, it becomes a sprout. The sprouts become canalized by 5 months. The primary channel forms the bases for the future teat cistern (Fig. 14-4).

Chronological development in bovines

Day 30, condensing ectodermal cells Day 35, mammary line Day 43, mammary bud Day 65, teat development Day 80, sprout Day 150, channel formation

Figure 14-1. Timeline for the development of the mammary gland in bovines

Figure 14-2. Development of the mammary buds in bovines

Figure 14-3. Conical mammary bud

Figure 14-4. Formation of the primary channel which will evolve into a cistern

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Subsequently, secondary cords grow to make the gland cistern and major ducts; there is some stroma deposited here giving the shape of the mature udder (Fig. 14-5).

All canalization is presently explained by apoptosis instead of remodeling or the migration of cells. This process is similar in the ewe and nanny but, in the sow and the mare there are two primary cords associated with each bud and there are no teat cisterns. There is little development until birth (Fig. 14-6).

Post-natal growth

After birth, there is little mammary development until 2 months before puberty. Whatever growth takes place in this period is isometric between the body and the mammary gland (Fig. 14-7).

As puberty approaches the influence of increasing concentrations of estrogens, in association with GH and glucocorticoids (Fig. 14-8), stimulates further duct development resulting in allometric growth, for the first couple of cycles. If pregnancy does not take place, then further growth returns to an isometric relationship with the body.

Once the animals start cycling, there is an associated cyclic change in epithelial tissue. The epithelium of the ducts becomes cuboidal during estrus and columnar during the luteal phase. Mammary growth is ovarian

Figure 14-5. Formation of secondary cords where the main ducts will develop and the cistern is formed

Figure 14-6. Separation of the structures that will eventually form each quarter

Body mammary growth

Birth to puberty isometric First few estrus allometric Until conception isometric Until parturition allometric Future lactations allometric

Figure 14-7. Growing mode of the mammary gland at different developmental stages

Figure 14-8. Development of intra-lobar ducts at the time the heifer reaches puberty. This is the result of elevation in estrogen, glucocorticoids and GH

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dependent, as demonstrated by comparing it to that of ovarioectomized animals, which reaches only 1/3 to 1/10 of that in an intact animal.

During early pregnancy, there is further duct development and growth, with branching into the interlobular areas and the alveoli start to appear (Fig. 14-9).

Towards mid pregnancy, a rudimentary system of the glandular lobes and lobules are well formed. By the end of pregnancy, most of the stroma is mainly connective tissue separating lobules or parenchyma separating lobes. The alveoli have become distended with milk secretion rich in fat globules and immunoglobulins. Contrary to previous belief, well-differentiated cells still retain the ability to proliferate even close to the onset of milk secretion. After lactation commences, the rate of cell proliferation is diminished with respect to the other developmental stages. Extra milk synthesis within 2 months of starting lactation results in higher milk production later on in lactation. Perhaps because more well-differentiated cells are recruited to produce milk as a result of either: improvement in the productive capacity of previously not well differentiated alveolar cells or; by higher proliferation of milk producing cells.

Lack of milk removal leads to the regression of the secretory tissue as a result of some secretory cells undergoing dedifferentiation and others undergoing apoptosis.

HORMONAL CONTROL OF MAMMOGENESIS The processes of mammogenesis or mammary development (Fig. 14-10), lactogenesis or initiation of milk production, and galactopoiesis or maintenance of milk secretion are strongly controlled by the endocrine system.

Role of ovarian steroids in mammogenesis

Ovarian activity is important in the development of the mammary gland. Ovarioectomized heifers have smaller mammary glands in volume and in weight than intact animals. Specifically, the parenchyma tissue is affected. The ovarian activity appears to mediate the actions of GH, specifically through changes in IGF-I. Treatment of non-pregnant heifers with estrogen stimulates proliferation of mammary epithelial cells. The initiation of ovarian activity in the heifer results in the allometric growth of the mammary tissue, with respect to the body, for a few cycles and then settles into isometric growth until conception takes place. During cyclic activity, there is no significant exposure to estrogens and progesterone together; growth does not start in earnest until both hormones are present at the same time in sufficient quantities. This takes place during late pregnancy when the CL produces large amounts of progesterone and the feto-placental unit generates elevated levels of estrogens. During pregnancy, Prl and PL

Figure 14-9. Development of intra-lobular ducts during gestation

Hormonal control of mammary gland (hypophysectomised

animals)

Estrogens Progesterone GH Placental lactogens Prolactin Glucocorticoids GH and PL induce alveolar growth Steroids without GH and PL do not

exert any effect

Figure 14-10. Hormones involved in mammary growth

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provide support for mammogenesis in some species, such as sheep and goats, but its importance in cattle appears to be minimal.

Injections of estrogens and progesterone to non-pregnant cows, in quantities sufficient to elevate circulatory concentration to pre-partum levels for seven days, resulted in induction of lactation in 70% of the animals and these reached production levels of 70% higher than in their normal volumes. This effect appears to depend, and correlate with Prl levels available.

Mammogenesis depends not only on hormonal concentration but also on receptor availability within the mammary tissue, as well as, the presence of transport proteins and intracellular lipids that are capable of making steroids unavailable to the tissues. These mechanisms appear to be responsible for the removal of the progesterone block and the induction of lactogenesis observed at parturition. Concentrations of progesterone receptors on mammary tissue are well correlated with lobulo-alveolar development.

Role of anterior pituitary hormones in mammogenesis

In murine species, Prl and GH play a major role in successful mammogenesis. Growth of lobulo-alveolar tissue is dependent on the presence of Prl. It has been shown that disruption in the Prl signal cascade, such as the signal transducers and activators of transcription (STAT5a), causes failure in lactation and impairs the development of terminal end buds. Since these structures are not present in cows, the effect of Prl in cattle is less important than in other species.

Role of other hormones in mammogenesis

As mentioned before, ovarian steroids, GH and Prl certainly stimulate mammogenesis but several other hormones play a permissive and supportive role in mammary growth (Figs. 14-10, 14-11). Placental lactogens, which has both Prl and GH like activity, is one of them. Adrenal gland hormones and thyroid hormones also support growth through their role in normal metabolism. Mammary duct development is impaired in hypothyroidism. Relaxin and the parathyroid hormone also support mammogenesis. A protein called parathyroid hormone-related protein (PTHrP) appears to influence mammary uptake of Calcium during lactation. Other potent

growth stimulators are the family of insulin-like growth factors. IGF, which are also produced locally in the mammary gland, seem to regulate cell growth, cell differentiation, cell function maintenance, and prevent apoptosis. Other compounds supporting cell development are epidermal cell factors (ECF), as well as, transforming growth factors (TGF-α), amphiregulin and several

heregulins or substances of the family of the ECF. Their presence in mammary tissue during mammogenesis and lactogenesis has been demonstrated but their specific role is not yet understood, although it is known that they trigger DNA synthesis in mammary tissue.

Fibroblast growth factors (FGFs) are about 20 small peptides with homologous aa sequences and a strong affinity for heparin and heparin-like glucosaminoglycans (HLGAGs) of the extracellular matrix (ECM). Thus, acting in a paracrine fashion to stimulate local epithelial cell proliferation and mammary gland morphogenesis.

In hypophysectomized, ovariectomized and adrenalec-tomized animals, induction of mammary development to the point of lactation can be successfully achieved by a combination of estrogens, progesterone, GH, Prl, and adrenocortical hormones. Growth hormone and PL induce lobular alveolar growth. Steroids without GH and / or PL have no effect on mammary development. Steroids sensitize mammary tissue by increasing receptors to hypophyseal hormones. Furthermore, estrogens by themselves seem to stimulate hypophyseal hormones. In sheep and goats, increased circulatory PL coincides with rapid lobulo alveolar proliferation during pregnancy. Apparently, PL are more important than GH or Prl. Growth hormones can be a substitute for PL due to their similar structure.

Induction of growth (Normal animals)

Estrogens alone induce alveolar growth

o Larger than normal alveoli Estrogen and progesterone induce

normal growth

Figure 14-11. Mammary growth can be induced artificially with exogenous hormones

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ARTIFICIAL INDUCTION OF MAMMARY GROWTH

Estrogens induce alveolar growth but the result is the formation of larger than normal alveoli; therefore, the overall secretory area per unit of volume is low (Fig. 14-11).

Estrogens plus progesterone induce formation of normal alveolar structure. Progesterone stimulates the develop-ment of alveolar structures but inhibits the secretory ability of alveolar cells. Goats treated with Es alone start producing milk faster after the treatment than goats treated with a combination of progesterone and estrogen. Nevertheless, the animals treated with estrogen and progesterone reach higher yields.

ENDOCRINE REGULATION OF LACTOGENESIS

For lactogenesis to take place there has to be a developed mammary gland and a limited differentiation of the secretory epithelium. These take place as pregnancy progresses. Then, during the periparturient period, there is completion of the cellular differentiation as the onset of milk production and secretion takes place. At the time of parturition, the mammary gland changes from a fast-growing non-secreting tissue to a slow highly secretory tissue to a non-growing type of tissue. This change is endocrine mediated (Fig. 14-12).

As parturition approaches, the levels of estrogen, progesterone, glucocorticoids, and PL are high and together they promote the growth of the mammary

tissue. At this time, the levels of Prl are low but starting to increase (Fig. 14-13).

Two hormones are crucial in lactogenesis. Glucocorticoids, which are responsible for the development of the rough endoplasmic reticulum of the secretory cells and Prl which promotes maturation of the Golgi apparatus and the formation of secretory vesicles (Fig. 14-14).

Furthermore, progesterone, which promotes general mammary growth with special emphasis in the alveolar tissue, inhibits epithelial alveolar secretory capacity. As the

Initiation of lactation

At parturition the mammary gland switches from a growing non secretory tissue to a secreting, non-growing tissue

Change is endocrine mediated

Figure 14-12. Lactogenesis

Figure 14-13. Endocrine changes at the time of parturition play a crucial role in the transition of the mammary gland from a growing organ to a secretory organ

Endocrine influences on lactogenesis

Glucocorticoids o Development of RER

Prolactin o Maturation of Golgi o Secretory vesicles

Progesterone o Promotes mammary growth

especially alveolar tissue o Blocks epithelial secretion o As P4 decreases, the block is

removed

Figure 14-14. Effect of different hormones in the initiation of milk production

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levels of progesterone decrease during the initiation of labour, the blocking effect of progesterone is removed allowing lactogenesis to commence. After parturition has taken place and the placenta is expelled, the concentration of estrogens and progesterone in circulation decreases rapidly. These changes can be directly attributed to the fact that the CL is no longer functioning and the feto-placental unit no longer exists. At the same time, the stimulation of the adrenal is significantly reduced and PL are no longer available since their source has been removed (Figs. 14-12, 14-15).

All these changes stop general mammary development. Corticosteroid binding globulin levels in circulation are also reduced after parturition, allowing more corticosteroids to be available to exert muscle catabolism; thus recruiting more energy and proteins to be used in lactation.

ENDOCRINE REGULATION OF GALACTOPOIESIS

Maintenance of lactation after lactogenesis requires continuous secretion of galactopoietic hormones, growth factors, and the regular removal of the milk from the udder. The most essential hormones are Prl, GH, glucocorticoids and T3 (Fig. 14-15). Prolactin is secreted immediately as a result of suckling or milking, This pattern is more evident in early lactation and declines thereafter. There is a high correlation between milk yield and prolactin

levels shortly after milking (5 min). The response to prolactin last only about one hour. Growth hormone or somatotropin is definetly a galactogogue. It increases milk production by promoting synthesis of lactose, fat and protein in the mammary gland. This translate in an increase in the persistence of lactation. This effect, as all effects of GH is dependent on the availability of adequate nourishment. If during lactation the cow is pregnant, the placental lactogen produced by the cow further supports the ongoing lactation.

Although, the pituitary is essential in the maintenance of lactation, the thyroid, parathyroid, and adrenals also influence lactation possibly through adequate maintenance of general metabolism. Milk production peaks at 9 weeks in cows and about 4 weeks in sows. This increase compared to the amount produced at parturition is mainly a consequence of enhanced secretory capabilities of the existing secretory mass, although some further cell differentiation continues to take place after parturition (Fig. 14-17).

Milk removal

In order to maintain milk production it is essential to remove the milk content from the mammary gland at frequen regular intervals. Ot as a hormone essential for milk removal is paramount to maintaining lactation because milk removal prevents secretory cell involution. Milk removal also removes a substance which temporarily inhibit milk secretion. This compound is named feedback inhibitor of lactation (FIL). It is produced in the milk secretory cells and inhibits, in an autocrine fashion, the production of further milk when the alveoli is filled with

Mammary growth slow down

Most hormones involved in growth have been removed o Progesterone

CL has regressed and placenta is removed

o Estrogens Feto-placental unit no

longer available o Placental lactogens

Placenta was expelled

Figure 14-15. After parturition mammary growth slows down because most growth promoting hormones are not longer available

Maintenance of lactogenesis

Responsibility of prolactin and growth hormone

Supported by thyroid, parathyroid and adrenal glands through adequate metabolic function

Removal of feedback inhibitor of lactation during milking

Figure 14-16. Hormones in charge of supporting continuous milk production

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milk. These factors and hormones that maintain alveolar cell population are important in total milk production and in determining the shape of the lactation curve. Not removing the milk wil result in increased mammary pressure that results ina reduction of of blood irrigation to the mammary tissue. Furthermore tha action of removing milk through suckling or mechanically stimulates prolactin production.

MILK SYNTHESIS AND SECRETION

Different components of milk are secreted by different mechanisms. So far, at least five routes of secretion used by different components of milk have been demonstrated (Fig. 14-18; 14-19).

Through the membranes Materials such as water, glucose, urea, and some ions from within the interstitial compartment can cross the basolateral membranes of the epithelial cells, move through the cells and cross the apical membrane towards the alveolar cavity where the milk accumulates.

Using the Golgi apparatus

Materials such as lactose, caseins, whey proteins, citrate and calcium, which are either synthesized within the cell or acquired from circulation, are then retained and packaged

into secretory vesicles and attached to the Golgi apparatus. These vesicles move towards the apical membrane and, either individually or in chains, fuse with the apical membrane and discard their contents into the lumen of the alveoli.

Using milk fat

Milk fat synthesized in the cell forms droplets that can transport fat soluble compounds, such as hormones and drugs. As the droplets are released in the apical membrane, small amounts of cytoplasm can leak into the lumen of the alveoli.

Figure 14-17. Lactation curves for a cow and a sow. Piglets are normally weaned before the lactation capabilities peak in the sow

Figure 14-19. Transport of materials from circulation, through the epithelial cells into milk, can be done through several routes

Milk synthesis

Figure 14-18. Possible mechanisms for milk synthesis

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Transcytosis mechanism

The basal membrane can form vesicles by either endocytosis or pinocytosis and the materials are transported in membrane-bound vesicles to the apical membrane where they are discarded by exocytosis. The contents are opportunistic and varied; this mechanism does not contribute a large amount to the milk volume.

Paracellular route

MILK REMOVAL

The neuroendocrine reflex that triggers milk expulsion has been exploited for over 4,000 years. This reflex can be caused by vaginal stimulation or mammary gland massage. The reflex consists of sending neural messages to the hypothalamus. This, in turn, immediately causes the release of pre-synthesized oxytocin into circulation. Once oxytocin comes in contact with tissues containing myoepithelial cells, with Ot receptors such as the mammary glands, it stimulates contraction of these cells. Since these cells are located around the alveoli they

force the milk out of the alveoli, through the intercalary ducts into the intralobular ducts (Figs. 14-20 to 14-25).

This mechanism involves the direct passage of materials from the interstitial fluid between the epithelial cells into milk. This mechanism is not of much importance but it can function in reverse. This explains why some of the milk components, such as lactose and whey proteins can be detected in circulation. This type of transport takes place in cases of inflammation such as during mastitis, as well as, when super-physiological doses of Ot are injected to trigger milk let down. The permeability of the epithelial lining to this mode of transport is significantly reduced around parturition, when the presence of glucocorticoids promotes the development of tight junction between epithelial cells.

Figure 14-21. Regulation of milk let down

Milk let down

Induced by tactile or conditioned stimulus, through oxytocy exerting their

effect on myoepithelila cells of the alveoli

Figure 14-20. Milk let down is mediated through oxytocin

Figure 14-22. Composed of epithelial secretory cells around a lumen which empties in the intercalary duct

Figure 14-23. A collection of alveoli makes up a lobule which empties through the intra-lobular duct to the inter-lobar duct

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Milking, be it mechanical or by suckling, triggers Prl secretion. It appears that the physical stimulation of the teat and not the removal of the milk causes the secretion of Prl. Mammary tissue secretes specific proteins in response to stimulation with Prl. Total milk production is influenced by Prl in many species but not much in cattle.

Stress inhibits milk secretion by causing peripheral vasocontriction and thus reducing the flow of blood containing Ot to the mammary gland. It is therefore important to minimize stress during the milking process. Since the sow does not have cisterns in the mammary gland, any impairment of Ot reaching the udder prohibits milk let down. On the other hand, the cow can

accumulate half of the milk production in the cistern, thus it is not as crucial to get this fraction of the milk out.

The mechanism for milk extraction exerted by the suckling calf does not consist exclusively in suction from the teat, but rather through massaging of the teat with the tongue exerting mechanics similar to those generated by manual milking. Milking machines remove milk by negative pressure in the teat; suction, with some lateral pressure, exerted via the lining of the milking cup facilitates venous return within the teat.

REGRESSION OF THE MAMMARY

Involution of the mammary gland involves apoptosis of alveolar cells. In small animals, such as rodents, this is a fast and significantly irreversible process. In large animals, involution is much slower and results in less loss of alveolar structure. Three days, after milking is stopped casein and α-lactoalbumin are reduced. Contrary to

previous beliefs, the alveolar tissue does not disappear completely from the mammary gland after prolonged periods without suckling or mechanical milking. Even 6 weeks after suspension of milking there is not a complete destruction of the alveolar tissue. Resumption of milking after 12 days without milk removal triggers re-differentiation of cells to a productive status.

Regression occurs slowly throughout lactation as a consequence of damage to alveolar cells, with a gradual decrease in milk production. If milk removal is stopped, the process of mammary regression can be induced rapidly. As milk accumulates in the alveoli, it distends the epithelium, creating overall pressure, which compresses the capillaries and decreases blood flow. This mechanical effect culminates with the rupture of some alveolar cells about 4 days after milk removal has stopped. This in turn triggers the release of the contents in the lysosomes, which help to digest the cellular material that is eventually reabsorbed (Fig. 14-26).

MASTITIS

Any inflammation of the mammary tissue is recognized as mastitis. The most common cause for mastitis is an invasion of the mammary gland, or a section of it by microorganisms (Fig. 14-27).

Figure 14-24. Several lobules make up a lobe which empties through the intra-lobar duct to the inter-lobar duct

Figure 14-25. Many lobes make up a quarter which empties in a cistern or to the teat

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The mammary gland is most vulnerable to microorganisms’ invasion during the beginning of the drying off period and before parturition. After drying off some cells regress and die, others are destroyed and most of the milk component has to be reabsorbed. This is carried out by neutrophils and macrophages, thus distracting these immune cells from attacking opportunistic bacteria, which then can establish colonies. During the peri-parturient period the concentration of glucocorticoids and estrogens are high, therefore, inhibiting immune response. A palliative approach commonly administered is to use long-lasting antibiotics treatment at drying off.

A common indicator of milk quality and udder health is the Milk Somatic Cell Count (MSCC). These represent normal cells of the body released into the milk and are mainly made of leucocytes (98% principally made of neutrophils and polymorphonuclear cells [PMNs]) and a small proportion of epithelial cells sloughed off into the lumen of the alveoli (Fig. 14-28).

Most uninfected milk contains less than 200,000 cells/mL, ideally less than 50,000 cells/mL. MSCC of 400,000 cells/mL reflects a minor inflammation, most likely caused by mastitis-producing organisms. Leucocytes are attracted to the milk by chemo tactic compounds released by the invading bacteria or by injured mammary cells. Enzymes released by PMNs can destroy secretory cells in localized areas and only a few of the cells are regenerated during the ongoing lactation, thus reducing the milk producing capacity of the mammary gland.

Furthermore, debris of cells and milk related proteins could cause localized blockades of ductules, thus, preventing the expulsion of milk from small areas or lobules within the mammary gland. As a result, in the long term, healthy cells in these blocked areas regress and dedifferentiate causing a further loss in milk producing capacity. Four forms of mastitis have been identified (Fig. 14-29).

Involution of the mammary gland

Apoptosis of alveolar cells Probably mediated by estrogens

o Decrease in late lactation Histological changes are noticeable

48 hr after milking has stopped o Vacuolation of epithelial cells

High intra mammary pressure o Reduces circulation o Destroys some cells

Figure 14-26. Mechanisms involved in the involution of the mammary gland

Mastitis

Inflamation of secretory tissue Most common

o Before parturition o At drying off

Associated with high MSCC

Figure 14-27. Characteristics of mastitis

Milk somatic cell count

Made of leucocytes (98%) o Neutrophils o Polymorphonuclear cells

Epithelial cells (2%) <50,000 - 200,000/mL uninfected milk MSCC of 400,000/mL reflects an

infection

Figure 14-28. Somatic cell count

Mastitis

Subclinical o Milk appears normal with elevated

MSCC Acute or clinical

o Swollen quarter, painful, hot o Fever and reduced appetite

Gangrenous o Cold bluish quarter

Chronic o Repeated clinical episodes

Figure 14-29. Most common types of mastitis

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Sub clinical

This is the most prevalent type of mastitis with an incidence of 15 to 40 cases of sub clinical mastitis for every clinical case detected. Milk in sub clinical mastitis appears normal but with an elevated MSCC. This can be identified with an on farm test such as the California Mastitis Test (Fig. 14-30) or the Wisconsin Mastitis Test. Usually, sub-clinical mastitis precedes a clinical case and in all cases results in decreased milk production.

Chronic mastitis

Chronic mastitis consists of repeated clinical episodes. This may eventually turn the mammary gland into a rigid, hard gland that ceases to respond to treatment even if the symptoms temporarily disappear. About 95% of the cases are produced by streptococci, staphylococci or coliforms. Less common agents are mycoplasma bovis, pseudomonas aeruginosa, actinomycis pyogens as well as some yeasts and moulds.

Acute or clinical mastitis

This is characterized by swelling of the affected quarters of the mammary gland, which is hot and painful to the touch. The animal with clinical mastitis may have a fever and reduced appetite. The milk will have visible clots and / or flakes and may be bloody. The quality of the milk is significantly reduced.

Gangrenous mastitis

It is characterized by causing the affected quarters to have a bluish discolouration and to be cold to the touch.

California Mastitis Test

Figure 14-30. The California mastitis test allows the identification of the status of each quarter of the animal.