**1.2. The ultimate determinants of milk production potential: mammary cell number and activity**

Milk production potential is a function of the number of mammary epithelial cells in the gland, as well as the secretory activity of those cells (Akers, 2002; Capuco et al., 2003; Boutinaud et al., 2004). Therefore, improved lactation performance can be achieved under conditions that enhance mammary cell proliferation (or decrease apoptosis), biochemical and structural differentiation of mammary epithelium, and synthesis and secretion of milk

© 2012 Wall and McFadden, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Wall and McFadden, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

components. Moreover, any factors involved in the regulation of these processes can directly impact mammary function and milk yield (Akers, 2002).

The majority (~80%) of mammary cells are formed during pregnancy and prior to lactation; however, cell proliferation during established lactation has been observed in both rodents (Tucker, 1969) and ruminants (Knight & Peaker, 1984; Capuco & Akers, 1990). Because the DNA content per mammary cell nucleus remains relatively constant during pregnancy and lactation, total DNA is considered an accurate indicator of mammary cell number (Tucker, 1987). Mammary cell secretory activity can be estimated by quantification of mammary RNA, and the ratio of RNA to DNA (Paape & Tucker, 1969). Measurements of both DNA and RNA content have provided insight into the relationship between mammary cell number, secretory activity, and milk yield.

It is well established that total mammary cell numbers and milk yield are positively correlated in both ruminants (Linzell, 1966; Keys et al., 1989) and rodents (Tucker, 1969; Nagai & Sarkar, 1978). The secretory activity of these cells is also an important factor involved in determining milk production potential. During lactogenesis, the mammary epithelium becomes highly differentiated. This period is associated with an overall increase in the size and metabolic activity of each cell, closure of tight junctions between cells, an increase in mitochondrial size, and development of the endoplasmic reticulum (Nickerson & Akers, 1984). During established lactation, any new cells that are formed are thought to become differentiated almost immediately (Tucker, 1969). The increase in milk yield during early lactation is associated with an increase in mammary DNA, followed by an increase in mammary cell secretory activity (Knight & Peaker, 1984). In addition, enhanced milk production potential is associated with increases in both mammary epithelial cell number and secretory activity (Keys et al., 1989). In rodents, successful rearing of pups and high rates of litter weight gain are both associated with an increase in mammary DNA, RNA, and ratio of RNA to DNA (Hackett & Tucker, 1969; Paape & Tucker, 1969). Consistent with the observed effects of mammary cell number and secretory activity on milk yield, the declining phase of lactation has been associated with losses in both mammary cell number and metabolic activity (Tucker, 1969; Knight & Peaker, 1984).

Taken together, these observations illustrate the importance of mammary cell number and secretory activity in determining milk yield. Therefore, to improve lactational performance of dairy cows, it is critical to understand the factors involved in the regulation of mammary development and differentiation. Indeed, novel management strategies based on discoveries in mammary gland biology have proven highly successful for use in improving milk production efficiency (Dunlap et al., 2000; Dahl et al., 2004). Some of these techniques will be discussed in more detail later in this chapter.

## **2. Hormonal regulation of mammary function**

One of the major roles of the endocrine system is to coordinate mammary function with the reproductive state of the animal. This physiological synchronization is a very complex process that involves the action and interaction of multiple hormones, as well as the interplay between hormones in the circulation and local regulation of the mammary response to these hormones. Although much of this chapter will be focused on local regulation of mammary function, it is important to appreciate the role of hormones in regulating mammary function and milk yield. During lactation, several key hormones are involved in the regulation of mammary cell number, secretory activity, and consequent milk production potential.

## **2.1. Hormones involved in lactogenesis and lactation**

## *2.1.1. Prolactin*

258 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

impact mammary function and milk yield (Akers, 2002).

metabolic activity (Tucker, 1969; Knight & Peaker, 1984).

**2. Hormonal regulation of mammary function** 

discussed in more detail later in this chapter.

number, secretory activity, and milk yield.

components. Moreover, any factors involved in the regulation of these processes can directly

The majority (~80%) of mammary cells are formed during pregnancy and prior to lactation; however, cell proliferation during established lactation has been observed in both rodents (Tucker, 1969) and ruminants (Knight & Peaker, 1984; Capuco & Akers, 1990). Because the DNA content per mammary cell nucleus remains relatively constant during pregnancy and lactation, total DNA is considered an accurate indicator of mammary cell number (Tucker, 1987). Mammary cell secretory activity can be estimated by quantification of mammary RNA, and the ratio of RNA to DNA (Paape & Tucker, 1969). Measurements of both DNA and RNA content have provided insight into the relationship between mammary cell

It is well established that total mammary cell numbers and milk yield are positively correlated in both ruminants (Linzell, 1966; Keys et al., 1989) and rodents (Tucker, 1969; Nagai & Sarkar, 1978). The secretory activity of these cells is also an important factor involved in determining milk production potential. During lactogenesis, the mammary epithelium becomes highly differentiated. This period is associated with an overall increase in the size and metabolic activity of each cell, closure of tight junctions between cells, an increase in mitochondrial size, and development of the endoplasmic reticulum (Nickerson & Akers, 1984). During established lactation, any new cells that are formed are thought to become differentiated almost immediately (Tucker, 1969). The increase in milk yield during early lactation is associated with an increase in mammary DNA, followed by an increase in mammary cell secretory activity (Knight & Peaker, 1984). In addition, enhanced milk production potential is associated with increases in both mammary epithelial cell number and secretory activity (Keys et al., 1989). In rodents, successful rearing of pups and high rates of litter weight gain are both associated with an increase in mammary DNA, RNA, and ratio of RNA to DNA (Hackett & Tucker, 1969; Paape & Tucker, 1969). Consistent with the observed effects of mammary cell number and secretory activity on milk yield, the declining phase of lactation has been associated with losses in both mammary cell number and

Taken together, these observations illustrate the importance of mammary cell number and secretory activity in determining milk yield. Therefore, to improve lactational performance of dairy cows, it is critical to understand the factors involved in the regulation of mammary development and differentiation. Indeed, novel management strategies based on discoveries in mammary gland biology have proven highly successful for use in improving milk production efficiency (Dunlap et al., 2000; Dahl et al., 2004). Some of these techniques will be

One of the major roles of the endocrine system is to coordinate mammary function with the reproductive state of the animal. This physiological synchronization is a very complex process that involves the action and interaction of multiple hormones, as well as the As the name indicates, prolactin (**PRL**) is known as the hormone of lactation. Accordingly, it has received much attention from lactation biologists studying the hormonal regulation of mammary function. In ruminants, PRL and glucocorticoids provide the primary stimulus for lactogenesis (Akers, 1985). A role for PRL in the onset of lactation was indicated by a peak in concentrations of the hormone in circulation immediately prior to parturition (Ingalls et al., 1973). Akers et al. (1981a; 1981b) used a dopamine agonist to inhibit periparturient PRL secretion in dairy cows, and this resulted in failure of the mammary epithelium to reach complete structural differentiation. The inhibition of cellular differentiation was accompanied with a 35% reduction in mammary RNA content, a decrease in rates of lactose and fatty acid biosynthesis, and a 40% reduction in milk yield (Akers et al., 1981a). In addition, cytological analysis revealed that inhibition of PRL secretion resulted in a decrease in the size of the metabolic machinery of the cell, including the rough endoplasmic reticulum and the Golgi Apparatus (Akers et al., 1981b). These effects were reversed by treatment with exogenous PRL; therefore, periparturient PRL secretion is essential for complete biochemical and structural differentiation of the mammary gland.

During established lactation, PRL is released during milking or suckling, indicating a role for the hormone in the maintenance of milk production (Koprowski & Tucker, 1973b; Akers, 1985). Indeed, PRL has been shown to maintain both the structural integrity and the functional activity of the mammary epithelium during lactation in rodents (Tucker, 1969; Flint & Gardner, 1994). In addition, PRL maintains and enhances lactation performance in rabbits (Cowie, 1969). Reports on the effect of PRL on milk yield in dairy cows, however, have been inconsistent (Plaut et al., 1987; Wall et al., 2006; Lacasse et al., 2008; Titus et al., 2008). It is generally accepted that PRL is not involved in galactopoiesis (the maintenance of milk production) in ruminant species (Tucker, 2000; Akers, 2006).

As mentioned previously, PRL and glucocorticoids are the major mediators of lactogenesis in many species. In addition to a well-established role in the structural differentiation of the mammary gland, PRL initiates lactogenesis by stimulating the mammary expression and secretion of milk proteins. Using explant culture, Guyette et al. (1979) reported that PRL and glucocorticoids stimulated the expression of casein mRNA within 1 hr of treatment. Similar observations were subsequently made for α-lactalbumin gene expression (Goodman et al., 1983). Subsequent research has confirmed that indeed, PRL and glucocorticoids elicit an

increase in mRNA and protein expression, as well as a decrease in the degradation of milk protein gene transcripts (Vonderhaar, 1987; Rosen et al., 1999). The ability of PRL and glucocorticoids to regulate casein gene expression is due to the presence of response elements in the promoter region of the casein gene (Rosen et al., 1986).

The action of PRL in the mammary gland is mediated through its receptor, which activates the Janus kinase/signal transducers and activators of transcription (**JAK/STAT**) pathway (Hennighausen et al., 1997a). Stimulation of casein and α-lactalbumin gene expression by PRL is mediated mainly by STAT5a, which is essential for both mammary gland development and lactation (Hennighausen et al., 1997b; Hynes et al., 1997; Horseman, 1999). Expression of the PRL receptor is also critical for normal mammary development and differentiation. In rodents, the number of PRL receptors in the mammary gland is positively correlated with milk yield and litter weight gain (Sakai et al., 1985). Additional evidence supporting a direct role for PRL receptor in the mammary gland was reviewed by Ormandy et al. (2003). The results of knockout experiments have revealed that there is a minimum requirement for PRL receptor expression in the mammary epithelium of mice, and this is critical for normal mammary development, lactogenesis, and lactation.

## *2.1.2. Glucocorticoids*

Cortisol is the main glucocorticoid in cattle, and, as mentioned previously, its major function is to enhance the action of PRL in stimulating differentiation of the epithelium and milk protein gene expression in the mammary gland during lactogenesis (Akers, 2002). In addition, glucocorticoids are involved in the regulation of tight junction closure (Stelwagen et al., 1998) and uptake of glucose by the mammary gland (Paterson & Linzell, 1974) during lactogenesis. In pregnant dairy cows, administration of exogenous glucocorticoids resulted in parturition and subsequent induction of lactation (Tucker & Meites, 1965).

During established lactation, glucocorticoids are released during milking or suckling in both rodents and ruminants (Koprowski & Tucker, 1973a; Ota et al., 1974). Interestingly, however, treatment with exogenous glucocorticoids is galactopoietic in rodents (Thatcher & Tucker, 1970) but not dairy cattle (Braun et al., 1970). It is thought that the galactopoietic effect of glucocorticoids in rodents is mediated via an increase in mammary cell secretory activity (Akers, 2002).

The action of glucocorticoids is mediated by its receptor, which is located in the cytosol of the mammary epithelial cell (Gorewit & Tucker, 1976). Upon binding to its receptor, the complex is translocated to the nucleus of the mammary epithelial cell, where it stimulates milk protein gene expression (Tucker, 1985; Li & Rosen, 1994). In addition, the glucocorticoid receptor has been observed to interact with PRL-activated STAT5 molecules to enhance the action of PRL in inducing β-casein gene expression (Wyszomierski et al., 1999). Surprisingly, however, local expression of glucocorticoid receptor was not critical for normal function during lactogenesis and lactation of mice (Kingsley-Kallesen et al., 2002). Therefore, although glucocorticoids enhance the action of PRL during lactogenesis, their direct action on the mammary gland is not essential for normal lactation in rodents. It is unknown whether expression of glucocorticoid receptor is required for normal mammary function during lactation of ruminants.

#### *2.1.3. Growth hormone*

260 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

elements in the promoter region of the casein gene (Rosen et al., 1986).

critical for normal mammary development, lactogenesis, and lactation.

in parturition and subsequent induction of lactation (Tucker & Meites, 1965).

*2.1.2. Glucocorticoids* 

activity (Akers, 2002).

increase in mRNA and protein expression, as well as a decrease in the degradation of milk protein gene transcripts (Vonderhaar, 1987; Rosen et al., 1999). The ability of PRL and glucocorticoids to regulate casein gene expression is due to the presence of response

The action of PRL in the mammary gland is mediated through its receptor, which activates the Janus kinase/signal transducers and activators of transcription (**JAK/STAT**) pathway (Hennighausen et al., 1997a). Stimulation of casein and α-lactalbumin gene expression by PRL is mediated mainly by STAT5a, which is essential for both mammary gland development and lactation (Hennighausen et al., 1997b; Hynes et al., 1997; Horseman, 1999). Expression of the PRL receptor is also critical for normal mammary development and differentiation. In rodents, the number of PRL receptors in the mammary gland is positively correlated with milk yield and litter weight gain (Sakai et al., 1985). Additional evidence supporting a direct role for PRL receptor in the mammary gland was reviewed by Ormandy et al. (2003). The results of knockout experiments have revealed that there is a minimum requirement for PRL receptor expression in the mammary epithelium of mice, and this is

Cortisol is the main glucocorticoid in cattle, and, as mentioned previously, its major function is to enhance the action of PRL in stimulating differentiation of the epithelium and milk protein gene expression in the mammary gland during lactogenesis (Akers, 2002). In addition, glucocorticoids are involved in the regulation of tight junction closure (Stelwagen et al., 1998) and uptake of glucose by the mammary gland (Paterson & Linzell, 1974) during lactogenesis. In pregnant dairy cows, administration of exogenous glucocorticoids resulted

During established lactation, glucocorticoids are released during milking or suckling in both rodents and ruminants (Koprowski & Tucker, 1973a; Ota et al., 1974). Interestingly, however, treatment with exogenous glucocorticoids is galactopoietic in rodents (Thatcher & Tucker, 1970) but not dairy cattle (Braun et al., 1970). It is thought that the galactopoietic effect of glucocorticoids in rodents is mediated via an increase in mammary cell secretory

The action of glucocorticoids is mediated by its receptor, which is located in the cytosol of the mammary epithelial cell (Gorewit & Tucker, 1976). Upon binding to its receptor, the complex is translocated to the nucleus of the mammary epithelial cell, where it stimulates milk protein gene expression (Tucker, 1985; Li & Rosen, 1994). In addition, the glucocorticoid receptor has been observed to interact with PRL-activated STAT5 molecules to enhance the action of PRL in inducing β-casein gene expression (Wyszomierski et al., 1999). Surprisingly, however, local expression of glucocorticoid receptor was not critical for normal function during lactogenesis and lactation of mice (Kingsley-Kallesen et al., 2002). Therefore, although glucocorticoids enhance the action of PRL during lactogenesis, their direct action on the mammary gland is not essential for normal lactation in rodents. It is Growth hormone (**GH**) is widely known for its galactopoietic effect in lactating dairy cattle. The first evidence of this was reported by Asimov and Krouze (1937), who observed that injections of dairy cows with pituitary extracts was associated with increased milk production. Although these findings represented an opportunity for increasing milk production efficiency of dairy cows, it was not practical to harvest and purify pituitary GH for commercial use until the 1980's, when the discovery of recombinant DNA technology made it possible to synthesize large quantities of GH. The recombinantly-derived bovine GH (**rbGH**) was subsequently used extensively for research, and was eventually approved for commercial use on dairy operations (Bauman, 1999). A galactopoietic effect of GH in rodents has not been observed (Tucker, 1985; Hadsell et al., 2007); however, Allan et al. (2002) suggested that GH is involved in maintaining mammary cell number during lactation of mice.

In ruminants, the action of GH on the mammary gland is thought to be mediated mainly by the insulin-like growth factor (**IGF**) signaling axis (Etherton, 2004). Treatment with exogenous GH increases the concentrations of IGF-1 in the circulation (Purup et al., 1993), which acts directly on the mammary gland (Shamay et al., 1988; Baumrucker & Stemberger, 1989). In addition to systemic IGF, locally produced IGF, as well as mammary expression of IGF receptor may influence mammary function and the response of the mammary gland to GH (Plath-Gabler et al., 2001; Akers, 2002). Indeed, the effect of GH on the mammary gland of ruminants varied with physiological state. During early lactation, treatment with exogenous GH had no effect on mammary cell proliferation in goats (Sejrsen et al., 1999). When administered during mid-lactation, however, GH was associated with an increase in mammary cell proliferation in cows (Capuco et al., 2001) and an increase in total volume of secretory tissue in goats (Knight et al., 1990). Because local production of IGF, as well as expression of IGF receptors are also physiologically regulated (Sinowatz et al., 2000; Plath-Gabler et al., 2001), this may explain the differences in the response to GH across physiological states.

Although the action of GH is mediated mainly through the IGF axis, there is evidence that GH may act independently of IGF-I to stimulate milk production (Hadsell et al., 2008). In addition, expression of GH receptor has been detected in mammary tissue (Knabel et al., 1998; Sinowatz et al., 2000; Plath-Gabler et al., 2001). The GH receptor belongs to a superfamily of transmembrane receptors, of which PRL receptor is a member (Postel-Vinay & Kelly, 1996). Therefore, the signaling pathway of GH is very similar to that of PRL: binding of GH to its receptor leads to activation of the JAK/STAT signaling pathway, which stimulates changes in gene expression in target tissues (Postel-Vinay & Kelly, 1996). Unlike PRL receptor, however, expression of GH receptor in mammary epithelium is not required for normal mammary development and function in rodents (Kelly et al., 2002). Instead,

expression of GH receptor in the mammary stroma is critical for normal mammary development, supporting the concept that the action of GH on the mammary epithelium is indirect and may be mediated by locally-produced IGF from the stroma (Kelly et al., 2002).

## **2.2. Other hormones**

## *2.2.1. Leptin*

Leptin is a hormone produced mainly by adipose tissue and is involved in appetite regulation. Although it is primarily associated with appetite regulation, leptin and its receptors are expressed in the mammary gland so it is thought to act locally to influence mammary development (Laud et al., 1999; Chilliard et al., 2001). Indeed, treatment of human mammary epithelial cells with leptin elicited a marked increase in cell proliferation (Hu et al., 2002). In contrast, treatment of bovine (Silva et al., 2002) or mouse (Baratta et al., 2003) mammary epithelial cells with leptin was associated with a decrease in proliferation. In fact, it is thought that leptin mediates the negative effects of a high-energy diet on mammary development of dairy heifers (Silva et al., 2002). In addition to the involvement of leptin in mammary development, it has also been proposed to work synergistically with prolactin to regulate mammary function and inflammation (Motta et al., 2004). More recently, it has been reported that leptin specifically induces expression of its long form receptor in goat mammary gland, and influences mammary development and function through several distinct JAK pathways (Li et al., 2010). Therefore, although the action of leptin in the mammary gland is not fully understood, it clearly plays a role in development and function and may directly influence changes in lactation efficiency by acting locally within the gland.

## *2.2.2. Melatonin*

Melatonin is secreted by the pineal gland during exposure to dark and is involved in the circadian rhythm of many biological functions. For over 30 years, melatonin has had an implicated role in mammary development due to its association with the incidence of breast cancer (Cohen et al., 1978). Indeed, a direct negative relationship between melatonin treatment or presence of the pineal gland and the development of mammary cancer was reported long ago (Tamarkin et al., 1981), and it has subsequently been well documented that melatonin inhibits mammary cancer (For reviews see Cos & Sanchez-Barcelo, 2000; Sanchez-Barcelo et al., 2003; Sahar & Sassone-Corsi, 2007; Pandi-Perumal et al., 2008). Because melatonin is secreted during the dark, and has a negative effect on breast cancer risk, the incidence of breast cancer is increased in night-shift workers and people with sleep disturbances (Stevens, 2006; Blask, 2009), and decreased in the blind (Feychting et al., 1998). It is thought that melatonin exerts its effects on breast cancer possibly by modulating estrogen receptor binding activity (Danforth et al., 1983; Cos et al., 2006; Hill et al., 2009).

Melatonin has also been observed to act directly on the mammary gland to inhibit growth in both rodents (Sanchez-Barcelo et al., 1990) and ruminants (Sanchez-Barcelo et al., 1991; Asher et al., 1994). As will be discussed in a later section of this chapter, exposure of lactating dairy cows to long day photoperiod (16h light; 8h dark) increases milk production, and exposure of late-pregnant cows to short day photoperiod (8h light; 16h dark) increases milk production in the subsequent lactation (Dahl et al., 2000; Dahl & Petitclerc, 2003). It was initially thought that this effect was mediated by melatonin. Because feeding melatonin did not mimic the effect, however (Petitclerc et al., 1998), alternative mechanisms have been proposed (Dahl, 2008). Nevertheless, melatonin plays a clear role in mammary development and function, and it may work together with other hormones, such as prolactin, to mediate the effects of varying daylength on milk production efficiency.

## *2.2.3. Oxytocin*

262 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

**2.2. Other hormones** 

*2.2.1. Leptin* 

within the gland.

*2.2.2. Melatonin* 

expression of GH receptor in the mammary stroma is critical for normal mammary development, supporting the concept that the action of GH on the mammary epithelium is indirect and may be mediated by locally-produced IGF from the stroma (Kelly et al., 2002).

Leptin is a hormone produced mainly by adipose tissue and is involved in appetite regulation. Although it is primarily associated with appetite regulation, leptin and its receptors are expressed in the mammary gland so it is thought to act locally to influence mammary development (Laud et al., 1999; Chilliard et al., 2001). Indeed, treatment of human mammary epithelial cells with leptin elicited a marked increase in cell proliferation (Hu et al., 2002). In contrast, treatment of bovine (Silva et al., 2002) or mouse (Baratta et al., 2003) mammary epithelial cells with leptin was associated with a decrease in proliferation. In fact, it is thought that leptin mediates the negative effects of a high-energy diet on mammary development of dairy heifers (Silva et al., 2002). In addition to the involvement of leptin in mammary development, it has also been proposed to work synergistically with prolactin to regulate mammary function and inflammation (Motta et al., 2004). More recently, it has been reported that leptin specifically induces expression of its long form receptor in goat mammary gland, and influences mammary development and function through several distinct JAK pathways (Li et al., 2010). Therefore, although the action of leptin in the mammary gland is not fully understood, it clearly plays a role in development and function and may directly influence changes in lactation efficiency by acting locally

Melatonin is secreted by the pineal gland during exposure to dark and is involved in the circadian rhythm of many biological functions. For over 30 years, melatonin has had an implicated role in mammary development due to its association with the incidence of breast cancer (Cohen et al., 1978). Indeed, a direct negative relationship between melatonin treatment or presence of the pineal gland and the development of mammary cancer was reported long ago (Tamarkin et al., 1981), and it has subsequently been well documented that melatonin inhibits mammary cancer (For reviews see Cos & Sanchez-Barcelo, 2000; Sanchez-Barcelo et al., 2003; Sahar & Sassone-Corsi, 2007; Pandi-Perumal et al., 2008). Because melatonin is secreted during the dark, and has a negative effect on breast cancer risk, the incidence of breast cancer is increased in night-shift workers and people with sleep disturbances (Stevens, 2006; Blask, 2009), and decreased in the blind (Feychting et al., 1998). It is thought that melatonin exerts its effects on breast cancer possibly by modulating estrogen receptor binding activity (Danforth et al., 1983; Cos et al., 2006; Hill et al., 2009).

Melatonin has also been observed to act directly on the mammary gland to inhibit growth in both rodents (Sanchez-Barcelo et al., 1990) and ruminants (Sanchez-Barcelo et al., 1991; Asher et al., 1994). As will be discussed in a later section of this chapter, exposure of Oxytocin is a peptide hormone that is secreted as part of the neuroendocrine response to milking or suckling (Goodman & Grosvenor, 1983). Once secreted into the bloodstream, oxytocin acts on the mammary gland to elicit the ejection of milk from the alveolar tissue so that it can be removed by the offspring or by the milking machine. Although it is mainly associated with milk ejection, treatment with exogenous oxytocin was associated with increased milk production of both dairy cows (Nostrand et al., 1991; Ballou et al., 1993; Lollivier & Marnet, 2005) and sheep (Zamiri et al., 2001). During milk stasis in lactating mice, treatment with exogenous oxytocin delays the onset of apoptosis and subsequent involution of the mammary gland (Akers, 1985). The action of oxytocin is mediated by its receptor, which is located on the membrane of myoepithelial cells in the mammary gland (Soloff, 1982; Reversi et al., 2005).

Local regulation of the response of the mammary gland to oxytocin has been observed. In lactating rats, milk stasis was associated with a decrease in the response of the mammary gland to exogenous oxytocin (Kuhn et al., 1973). Similarly in dairy cattle, Linnerud et al. (1966) observed that treatment with exogenous oxytocin did not increase milk yield in the absence of milk removal. In addition to the effects of mammary fill with milk, locally produced hormones are thought to influence the effects of oxytocin on the mammary gland of ruminants (Peaker et al., 1995).

#### *2.2.4. Ovarian hormones*

Estrogen and progesterone are both secreted by the ovary, as well as the placenta of pregnant animals, and these hormones are mainly involved in the growth and development of the mammary gland during puberty and pregnancy (Erb, 1977; Schams et al., 1984; Tucker, 1985). Both hormones, however, have additional roles during lactogenesis and lactation. Prior to parturition, estrogen is one of the first hormones to increase in circulation, indicating a role for estrogen in lactogenesis (Akers, 2002). Indeed, administration of exogenous estrogen has been used to induce lactation in both pregnant and non-pregnant dairy cattle (Meites, 1961; Smith & Schanbacher, 1973; Howe et al., 1975; Collier et al., 1977). Estrogen also stimulates the anterior pituitary gland to secrete PRL, and it increases the expression of PRL receptors in the mammary epithelium (Tucker, 2000). During established lactation, estrogen decreases milk yield by interfering with milk ejection (Bruce & Ramirez,

1970), and by inducing mammary involution (Athie et al., 1996; Bachman, 2002). Similar to GH, the action of estrogen on the mammary epithelium is thought to mediated locally by the mammary stroma and by local production of growth factors (Imagawa et al., 2002; Cunha et al., 2004; Parmar & Cunha, 2004).

Prior to parturition in dairy cattle, progesterone inhibits the synthesis of α-lactalbumin, casein, and lactose and consequently inhibits the onset of lactogenesis (Goodman et al., 1983; Wilde et al., 1984; Akers, 1985; Tucker, 2000). Once lactation has been established, however, progesterone has no effect on mammary function or milk production, probably because expression of progesterone receptor in lactating mammary gland is very low (Tucker, 2000).

#### *2.2.5. Relaxin*

Relaxin is a protein hormone that is involved in relaxing the pelvic ligaments around the time of parturition of several species (Sherwood et al., 1993). Although not classically considered to be involved in mammary development, research has shown that it is critical for normal mammary development in rodents (Bani et al., 1986), ruminants (Cowie et al., 1965), and pigs (Hurley et al., 1991; Bagnell et al., 1993). Relaxin is also thought to be involved in the inhibition of lactation prior to parturition (Harness & Anderson, 1975). Wahab and Anderson (1989) suggested that relaxin works synergistically with estrogen and progesterone to stimulate mammary growth in pregnant rats, and similar observations have been made in pigs (Winn et al., 1994). In mice, however, relaxin appears to work independently of sex hormones to stimulate nipple development (Kuenzi et al., 1995). Of particular relevance to milk production efficiency, the stimulus of suckling by piglets appears to overcome any effects of relaxin deficiency on lactation performance of lactating pigs (Zaleski et al., 1996). In mice, however, deletion of the relaxin gene resulted in death of pups due to insufficient nipple development and the inability of the pups to suckle (Zhao et al., 1999). Therefore, although relaxin clearly plays a role in mammary development and function, it is still unclear what role, if any, it plays during lactation. In addition, there are clear differences in the role of relaxin across species.

## *2.2.6. Thyroid hormone*

Thyroid hormones have no clearly established role in mammogenesis (Tucker, 2000), but they are galactopoietic in dairy cows. In addition, they may enhance the effect of other lactogenic and galactopoietic hormones such as PRL and GH (Capuco et al., 1989; Akers, 2002). Leech (1950) investigated the effects of exogenous thyroxine on milk yield of dairy cows, and reported that the hormone was galactopoietic in a dose-dependent fashion. The author speculated that thyroxine functions to increase mammary cell secretory activity; however, the treatment only transiently increased milk yield and upon cessation of treatment, milk yield decreased below pre-treatment levels (Leech, 1950). Consequently, although the milk yield response was investigated further (Stanley & Morita, 1967; Schmidt et al., 1971), treatment with exogenous thyroid hormone to increase milk production of dairy cows was never adopted by the dairy industry.


**Table 1.** The role of various hormones on mammary function during lactation

Clearly, the endocrine system plays an important role in the regulation of mammary function and milk yield across many species. In addition, there is substantial evidence for local regulation of the response of the mammary gland to the endocrine system. This local regulation includes changes in the expression of specific hormone receptors in the gland, as well as the local production of growth factors that mediate or enhance hormonal effects on mammary function. Moreover, there are regulatory mechanisms in the mammary gland that are thought to act separately from, and may sometimes interact with, the effects of the endocrine system.
