Mammary involution and relevant udder health management in sheep

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Mammary involution and relevant udder health management in sheep I.G. Petridis*, G.C. Fthenakis Veterinary Faculty, University of Thessaly, 43100 Karditsa, Greece

ARTICLE INFO

ABSTRACT

Keywords: Apoptosis Defence mechanisms Dry-period Ewe Goat Intramammary treatment Involution Mammary involution Mastitis Sheep

Objective of the paper is to describe mechanisms leading to mammary involution in ewes and changes taking place in the mammary gland during that period. Mammary involution is the regression of mammary tissue to non-secreting state and takes place as initiated, gradual or senile. In mutton-type production systems cessation of lactation is abrupt, whilst in dairy-type production systems it is progressive or abrupt. The period from cessation of milk removal until the beginning of subsequent lactation period is termed ‘dry-period’ and is distinguished into stage of active involution, stage of the ‘steady-state’ involution and stage of redevelopment and lactogenesis. The ‘dry-period’ is important in health management of sheep for optimum milk production during the subsequent lactation period, as it is necessary for renewal of mammary epithelial cells. Mammary involution is influenced by decreased activity of galactopoietic hormones and local mechanisms in response to milk accumulation in the gland. Milk accumulation plays a major role in triggering apoptosis of epithelial cells. There are two pathways leading to apoptosis, an intrinsic and an extrinsic. During involution, significant histological changes take place in the mammary gland, mainly reduction (up to 3.6% up to the 4th day) of the epithelial area of the gland and increase of its stromal part. The mammary gland is particularly susceptible to infections after cessation of lactation; progressively, that changes; a keratin plug is formed at the teat orifice, leucocytes accumulate in the gland and concentrations of immunoglobulins and lactoferrin increase. Udder health management at the end of a lactation period aims to cure infections that have occurred during the previous lactation period and prevent new intramammary infections during the ‘dry-period’. Culling ewes with at least one mammary gland permanently damaged or ones chronically affected or others with incidents of relapsing mastitis or not fully respondent to mastitis treatment during the preceding lactation period contributes to decrease of veterinary expenses for mastitis control in the flock, elimination of sources of potential infection for other animals in the flock and decrease of flock bulk somatic cell counts in the subsequent lactation period. Culling should be complemented with intramammary antibiotic administration at the end of a lactation period. In many clinical studies from around the world, administration of antimicrobial agents at the end of a lactation period has been found beneficial. Antibiotic administration at drying-off may be performed to all animals in a flock (‘complete’) or only to those considered to be infected (‘selective’). In all cases, maintenance of the prescribed withdrawal periods is essential to safeguard public health. The procedure should always be applied as part of a strategic udder health management plan in a flock; implementation improves the welfare of animals and affords significant financial benefits to the farmer. Correct udder health management in ewes at the end of a lactation period will contribute to improved mammary health for the forthcoming lactation period.

1. Introduction and definitions The mammary gland fulfils a variety of physiological, immunological and biochemical functions (Oliver and Sordillo, 1989). As part of the reproductive system of mammals, it undergoes repeated cycles of structural development, functional differentiation and regression (Hurley and Loor, 2011). During these cycles, mammary glands of adult animals undergo the following three distinct functional transitions. ⁎

• From end of lactation to lactogenesis, leading to regression of mammary tissue to non-secreting state, which is termed ‘involution’. • From lactogenesis (including the colostrogenesis) to start of a lactation period. • From start of a lactation period to beginning of involution. During each of the above, marked changes occur in size, structure and function of the organ. Moreover, distinct changes also occur in mammary secretion during each of the above physiological transitions

Corresponding author. E-mail address: [email protected] (I.G. Petridis).

https://doi.org/10.1016/j.smallrumres.2019.07.001 Received 31 March 2019; Received in revised form 29 June 2019; Accepted 1 July 2019 0921-4488/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: I.G. Petridis and G.C. Fthenakis, Small Ruminant Research, https://doi.org/10.1016/j.smallrumres.2019.07.001

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(Oliver and Sordillo, 1989). For mammary involution, the following three different procedures have been described (Hurley, 1989).

mammary cell renewal cannot be reached at short periods (BernierDodier et al., 2011). In studies, which had compared effects of type of involution process (i.e., initiated or gradual involution) on incidence risk of subsequent intramammary infections and mastitis development, no differences were evident between the two types of involution procedure (Petridis et al., 2012, 2013).

• Induced or initiated involution (occurring after abrupt cessation of milk removal). • Progressive or gradual involution (occurring during the declining phase of milk production during a lactation period). • Senile involution (occurring at end of the reproductive life of the

3. Physiological mechanisms involved in mammary involution

animal).

Active involution starts with the cessation of milk removal and continues for 3 to 4 weeks. It is followed by the ‘steady-state’ involution, at which stage the mammary gland is fully involuted. After involution, 3–4 weeks before the expected lambing date, starts the redevelopment of the mammary gland, with formation of the colostrum (colostrogenesis) (Hurley and Loor, 2011). In general, mammary involution is not regulated by the decreased activity of galactopoietic hormones (e.g., prolactin, growth hormone), but by the interruption of release of these hormones that follows cessation of milk withdrawal from the mammary gland. Local mechanisms occurring in response to milk accumulation in the mammary gland also play a significant role in the process of involution (Wilde et al., 1997, 1999). The volume of mammary secretion is greatly reduced during involution, with concurrent changes in concentrations of its constituents; for example, fat, protein and lactose concentrations decrease rapidly, whilst concentration of lactoferrin increases (Hurley and Loor, 2011; Hurley, 1989). Moreover, during active involution, there is increased leucocytic infiltration of the mammary parenchyma; the leucocytes participate in the involution process, specifically in the removal of fat globules, casein micelles and cellular debris (Tatarczuch et al., 2000, 2002).

In farm animals, initiated involution and gradual involution are, of course, those with highest importance. In these species, mammary involution occurs at the end of each lactation period and is characterised by reduction of numbers of mammary epithelial cells coupled with extensive proteolytic degradation of extracellular matrix (Quarrie et al., 1996; Flint et al., 2005). The period from complete cessation of milk removal until beginning of the subsequent lactation period is termed ‘dry-period’. The ‘dryperiod’ is distinguished in the following three distinct stages.

• Stage of active involution • Stage of the ‘steady-state’ involution. • Stage of redevelopment and lactogenesis (including colostrogenesis).

The ‘dry-period’ is of importance in health management of sheep for optimum milk production during the subsequent lactation period (Contreras et al., 2007; Fthenakis et al., 2012), as it is necessary for renewal of mammary epithelial cells (Capuco et al., 1997). Increased milk production in the subsequent lactation period is indicated by number of mammary epithelial cells, which depends on their proliferation and apoptosis rates (Knight, 2000; Capuco et al., 2003), and secretory activity of mammary epithelial cells, which depends on their differentiation (Akers et al., 2006). With no sufficient cell renewal after involution, milk yield produced during the subsequent lactation period would be declining fast, as a result of decrease in cell numbers and cellular secretory activity (Capuco and Akers, 1999). Moreover, intramammary infections during that period affect normal pre-partum development of mammary epithelial cells and subsequently lactogenesis (Oliver and Sordillo, 1989), hence, quality and quantity of milk to be produced. Objective of the present review is to describe the mechanisms which lead to mammary involution in ewes and the changes which take place in the mammary gland of ewes during that period, as well as to appraise udder health management in ewes at the end of a lactation period.

3.1. Mechanisms involved in involution of mammary epithelial cells Milk accumulation plays a major role in triggering apoptosis and, thus, involution of mammary epithelial cells (Green and Streuli, 2004). Among the various mechanisms proposed, one is the accumulation of pro-apoptotic factors (e.g., α-lactalbumin), which act through apicallylocated receptors, but during lactation are continuously removed at milking (Hakansson et al., 1995, 1999; Green and Streuli, 2004). Another factor may be the change in the shape of mammary epithelial cells by stretching, when alveolar lumen is engorged with milk accumulation after cessation of lactation. Preservation of the structure of mammary epithelial cells is important for their proper functioning; alteration of their shape may lead to damage in the tight junctions, resulting in the leakage of pro-apoptotic factors from the apical to the basal surface, where they would trigger apoptosis, either directly or by antagonising survival signals (Stelwagen et al., 1994, 1995, 1997; Singh et al., 2005). Moreover, stretch receptors could be activated (e.g., adhesion receptors linking the basal epithelial surface to the basement membrane) or cell to cell adhesion junctions may be damaged; both pathways lead to production of pro-apoptotic signals (E-cadherin) (Boussadia et al., 2002; Wernig et al., 2003; Green and Streuli, 2004). It has been shown that there is a decline in gene expression of various integrins, which mediate survival signals from the extracellular matrix to the mammary epithelial cells (Singh et al., 2005). Thus, it has been concluded that communications between mammary epithelial cells and the extracellular matrix would be compromised during the involution process and that there may be an interaction between integrins and growth factor receptors, since the integrins mediate the survival signals via the PI3-K/Akt survival pathway (Farrelly et al., 1999; Singh et al., 2005). Another regulator of apoptosis is the feedback inhibitor of lactation (FIL), a milk-whey protein with molecular mass of 7600 Da, which can reduce the rate of milk secretion, by inhibiting transfer of proteins through the Golgi apparatus within the mammary epithelial cells (Peaker and Wilde, 1996; Knight et al., 1998). Its concentration

2. General approach to mammary involution in sheep In flocks of dairy production system, mammary involution is either initiated (i.e., milking is stopped at once) or gradual (i.e., milking frequency is progressively decreased over a period of several days or weeks) (Petridis et al., 2012, 2013). In flocks of meat production system, mammary involution is initiated, as cessation of lactation takes place when lambs are removed from their dam (Sargison, 2008). In the para-Mediterranean areas (and other areas of the world in same latitude), often the stage of active involution may coincide with the initial stage of pregnancy. In North Europe (and other areas of the world in same latitude), usually mammary involution has been completed before mating and pregnancy. ‘Dry-period’ duration of at least 60 days is recommended for optimum involution and subsequent lactogenesis (Lee and Lascelles, 1969; Tatarczuch et al., 1997, 2000). A shorter ‘dry-period’ has been associated with incomplete renewal of mammary cells (Capuco et al., 1997) and decreased subsequent milk yield (Pinedo et al., 2011; Hernandez et al., 2012), because, perhaps, a hormonal environment suitable for 2

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increases with longer periods of milk accumulation, which way downregulating milk production in a chemical feedback loop (van Veldhuizen-Staas, 2007). FIL acts within the alveolar tissue, more precisely, in the apical surface of the secretory cells (Peaker and Wilde, 1996; Knight et al., 1998). The final consequences of blockage of the secretory pathway within the epithelial cells are the inhibition of protein synthesis and the down-regulation of the mammary prolactin receptors. The latter may render FIL a possible regulator of apoptosis, although this regulation would be more pronounced in gradual involution, with long intervals between milk removals, as induction of apoptosis seems to be a rather long term and indirect effect of the hormone’s action (Peaker and Wilde, 1996; Knight et al., 1998; Wilde et al., 1999). Mammary cell proliferation, differentiation and apoptosis are controlled by hormones and factors acting systemically (growth hormone, prolactin) or at mammary gland level (FIL, insulin-like growth factor 1 [IGF-1], insulin-like growth factor-binding protein [IGFBP]). The balance between cell proliferation and apoptosis defines the cell turnover in the mammary gland. Mammary epithelial cells are removed through increased apoptosis during active involution in a period of 3–4 weeks, whilst increased cell proliferation takes place at the stage of redevelopment and lactogenesis, continuing during the early stage of a lactation period. Survival of mammary epithelial cells is primarily dependent on the activity of IGF-1, which, in turn, is modulated by the production, locally, of IGFBPs from the stromal cells that surround them. Increased concentration of specific IGFBPs produced at the early stages of involution inhibit activity of IGF-1 and prevent it from binding to its receptors, which would suppress apoptosis and would delay involution (Wilde et al., 1999; Colitti and Farinacci, 2009; Hurley and Loor, 2011).

Korsmeyer, 2004). However, better understanding led to hypothesising new models. Most recently, it has been proposed that, under nonapoptotic conditions, a balance between pools of Bax in cytosol and mitochondria is maintained by the retro-translocation of anti-apoptotic proteins (Bcl-xL), although during apoptosis this balance is tipped and Bax is inserted in the outer mitochondrial membrane, leading to changes in its permeability (Colitti, 2012). This results to release of several pro-apoptotic molecules (e.g., cytochrome C, apoptosis inducing factor [AIF]) through the inter-membrane space. In the cytoplasm, cytochrome C binds to apoptosis activating factor-1 and forms the apoptosome, which further activates caspase-9 and leads to activation of executioner caspases and, at the end, induction of apoptosis. Additionally, AIF, a flavoprotein, normally confined to mitochondria, when stimulated by apoptotic factors translocates to the cytosol and nucleus, where it activates endonucleases that involve DNA fragmentation and changes mitochondrial membrane permeability (Antonsson, 2004; Colitti et al., 2004a, 2004b; Green and Streuli, 2004; Flint et al., 2005; Myers and McGavin, 2007). A growth factor with significant importance in survival signaling in the mammary gland is IGF-1. This is secreted primarily in the liver as an endocrine hormone and at various other tissues (among them, the mammary gland stroma) in a paracrine/autocrine way, stimulated by the growth hormone (Flint et al., 2005). IGF-1 stimulates mammary epithelial cell proliferation and inhibits apoptosis (Flint et al., 2005). Moreover, prolactin inhibits secretion of IGF-1 binding protein-5 (IGFBP-5) from mammary epithelial cells, enhancing that way action of growth hormone, since IGFBP-5 inhibits action of IGF-1. Hence, growth hormone is considered to be a factor promoting milk secretion indirectly, through stimulation of IGF-1 production, while prolactin acts directly in mammary epithelial cells (Flint et al., 2005; Colitti and Farinacci, 2009). IGF-1 activates several kinases, such as the phosphatidyl-inositol 3kinase, which in turn activates protein kinase B (Akt). This catalyzes phosphorylation of pro-apoptotic Bad and of forkhead transcription factor 3A (FOXO3A), preventing death of mammary epithelial cells (Green and Streuli, 2004; Colitti and Farinacci, 2009). FOXO3A upregulates a number of target genes in the nucleus, which promote cell death (Greer and Brunet, 2005; Wu et al., 2006); Bad normally sequesters anti-apoptotic agents (e.g., Bcl-2 or Bcl-x). Hence, in the presence of excess IGFBP-5, action of IGF-1 is inhibited, the pathway PI3K/Akt is blocked, leading to dephosphorylation of FOXO3A and Bad and subsequent up-regulation of cell death-promoting genes in the nucleus and decrease of the anti-apoptotic agents Bcl-2 and Bcl-x, at the end resulting to apoptosis. In the mammary glands of ewes, expression level of IGFBP-5 is up-regulated during involution (Colitti and Farinacci, 2009), perhaps stimulated by milk accumulation therein (Green and Streuli, 2004). An interesting finding from experimental work performed in murine mammary cell cultures is that mammary epithelial cells depend on the basement membrane for survival, because the IGF-1 signaling is more effective through the laminin-rich basement membrane than through the stromal extracellular matrix, which contains collagen I. As signal transduction is coordinated by integrins (receptors that link extracellular matrix-proteins to the cytoskeleton), this variation in efficiency might be due to the fact that different integrins are utilised for adhesion of mammary epithelial cells to proteins of basement membrane and collagen I. The interaction between integrins and basement membrane constitutes an important survival axis for mammary epithelial cells; a change in this interaction might activate secretory cell apoptosis (Pullan et al., 1996; Green and Streuli, 2004). Nevertheless, potential significance of the finding in ewes remains unclear, as in previous work performed specifically in that species (Tatarczuch et al., 1997; Colitti et al., 1999), no disruption of the basement membrane during involution has been observed. These differences between sheep and mice may possibly be explained by the existence of protease-independent and -dependent stages of involution of the mammary tissue and the

3.2. Pathways to apoptosis of mammary epithelial cells There are two major pathways leading to apoptosis, an intrinsic and an extrinsic. The intrinsic (or mitochondrial) pathway involves the mitochondria and can be triggered by various intracellular stressors, which change the integrity of the outer mitochondrial membrane. The extrinsic (or receptor-initiated) pathway involves death receptors of the cell surface and their activation by their ligands. These two mechanisms occur independently of each other and involve distinct molecular interactions (Green and Streuli, 2004; Myers and McGavin, 2007). Although they may interconnect and overlap at numerous steps (Green and Streuli, 2004), no shift between the two pathways during a mammary gland cycle has been found in cows (Norgaard et al., 2008). Induction of apoptosis is a multi-step process, where several pathways are involved. Some of these are pro-apoptotic, whilst others inhibit existing cellular survival pathways (Green and Streuli, 2004). 3.2.1. Intrinsic pathway In the intrinsic pathway, increase in the permeability of the outer membrane of mitochondria and the release of pro-apoptotic molecules (e.g., cytochrome C) in the cytosol trigger the beginning of apoptosis. Mitochondria act as stress sensors, which detect cell damage and changes in the environment, like withdrawal of survival signals. These survival signals and growth factors stimulate production of anti-apoptotic Bcl-2 family proteins (e.g., Bcl-2, Bcl-x) (Myers and McGavin, 2007). The family of the Bcl-2 proteins plays a key role in this pathway. Some members of the family (e.g., Bcl-2, Bcl-x) have anti-apoptotic activities, whilst others (e.g., Bad, Bak, Bax) have pro-apoptotic activities. The pro-apoptotic proteins are found in the cytosol, acting like sensors of cellular damage or stress, while the anti-apoptotic mainly reside on the surface of mitochondria. First, it was suggested that the fate of the cell was dictated by the balance between pro- and antiapoptotic proteins (‘rheostat’ model) and that the basic function of antiapoptotic proteins was binding free Bax molecules (Danial and 3

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initiation of the involution process, as discussed in detail by Colitti et al. (1999) Finally, another possible regulator of apoptosis is the leucaemia inhibitory factor (LIF), through activation of STAT3 pathway (Green and Streuli, 2004), the apoptotic target of which seems to be IGFBP-5 (Chapman et al., 1999). LIF activates the STAT3 pathway, causing upregulation of suppressor of cytokine signaling 3 (SOCS3), which attenuates the STAT5 pathway and feedback regulates the STAT3 pathway. (Singh et al., 2016). The balance between STAT5 and STAT3 signaling regulates transition from lactation to involution. Activation of STAT5 suppresses STAT3-mediated apoptosis and, thus, acts as a survival signal in mammary epithelial cells (Clarkson et al., 2006). The involution process is still reversible until up to 192 h after cessation of milking, as mammary epithelial cell are in a quiescent and not senescent phase yet (Singh et al., 2016).

degradation (Flint et al., 2005; Norgaard et al., 2008). 3.4. Changes in blood flow Diametre of the external pudendal artery, as assessed by ultrasonographic examination, progressively decreased after involution and became minimal after 37 days subsequently to initiation of involution (Petridis et al., 2014). Decrease of the diametre of the external pudendal artery contributes to the reduced amount of blood transferred to the mammary gland and that way contributes to the involution of the mammary tissue. Both resistance index and pulsatility index progressively increased during the involution process, obviously as the result of decreased blood perfusion as involution advanced (Petridis et al., 2014). Reduction of blood flow in the mammary gland may be a consequence of reduced blood requirement, due to the reduced milk secretory activity rather than of increased intramammary pressure, because of accumulation of milk after cessation of milk removal (Knight et al., 1998). Decreasing blood volumes provide weaker signals for Doppler spectrum analysis, because of the smaller number of moving erythrocytes (Widder and Goertler, 2004). Reduced blood flow may contribute to the increased susceptibility of involuting mammary glands, as smaller amounts of leucocytes would be transferred to the mammary gland.

3.2.2. Extrinsic pathway As observed in cows, the extrinsic pathway is initiated when death receptors are cross-linked by their ligands. The most commonly described death receptor present in the mammary gland is Fas. The interaction of FasL (Fas ligand) with Fas brings together three or more molecules of Fas (trimerisation), this way creating in the cytoplasm a binding site for an adapter protein called Fas associated death domain (FADD). Binded FADD results in activation of caspase-8 and consequent downstream activation of execution caspases (Green and Streuli, 2004; Myers and McGavin, 2007). However, no relevant data are available regarding the involution process specifically in ewes.

4. Changes in the structure of involuting mammary glands 4.1. Histological changes

3.3. Mechanisms involved in extracellular matrix remodeling

The initial change in involuting mammary glands is the formation of large stasis vacuoles in the epithelial cells, as a result of accumulation of secretory vesicles and milk fat globules in the cytoplasm of the alveolar cells. This accumulation is the result of reduction in fusion of secretory vesicles with the apical membrane and indicates that milk secretion ceases prior to milk synthesis inhibition (Hurley, 1989). As a consequence of decreased synthetic activity, a marked reduction in cytoplasmic organelles of epithelial cells involved in synthesis of milk components occurs already on the 2nd day of involution (Holst et al., 1987). Their number becomes minimal after four weeks subsequently to the cessation of milk removal (Holst et al., 1987; Sordillo and Nickerson, 1988). In sheep, the epithelial cells become flattened two days after cessation of milk removal, with simultaneous formation of large stasis vacuoles. As apoptosis advances, their cytoplasm fills with vacuoles with clusters of dense material and fragmented nucleus. By the 4th day, they fill with large empty vacuoles and autophagosomes in their cytoplasm and later, by the 7th day, they become highly vacuolated with small dense mitochondria, some rough endoplasmic reticulum and numerous free ribosomes. On the 30th and 60th days after cessation of milk removal, the epithelial cells have been found to be cuboidal with numerous ribosomes, mitochondria, a small Golgi apparatus and a small amount of rough endoplasmic reticulum, i.e., a characteristic image of a resting gland (Tatarczuch et al., 1997). The alveolar lumen initially becomes distended, but, from the 4th day after cessation of lactation, progressively, its size is reduced; 30 days after that, the alveoli appear to have little or no lumen. Ultimately, alveoli appear to be irregularly shaped and collapsing, shrunken or fully collapsed. The few epithelial cells in each alveolus are flattened and slender (Tatarczuch et al., 1997; Colitti et al., 1999). No lacteal content is present in the alveolar lumen. Nevertheless, the alveolar structure is maintained throughout the duration of involution (Capuco et al., 1997), as destruction of the mammary alveoli is not complete (Norgaard et al., 2008). Thirty days after initiation of the process, there is a marked reduction of numbers of the organelles involved in milk synthesis (rough endoplasmic reticulum, Golgi system), although sufficient numbers of mitochondria and ribosomes are still present in the cytoplasm of

In general, during involution, the stromal area increases, whilst during redevelopment of the mammary gland it decreases (Capuco et al., 1997; De Vries et al., 2010). This remodeling is partly regulated by the matrix metalloproteinases, which are endopeptidases secreted as pro-enzymes and when activated in the extracellular environment by various factors; for example, members of the plasminogen activator system degrade the extracellular matrix proteins. In this system, plasminogen, the inactive zymogen precursor of plasmin, is activated by urokinase-type plasminogen activator and tissue-type plasminogen activator. This activation can be inhibited by the plasminogen activator inhibitor. Except from activating matrix metalloproteinases, those proteases are also involved in direct degradation of extracellular matrix. The action of matrix metalloproteinases is controlled to prevent inappropriate degradation or proliferation of mammary tissue, by gene transcription, the aforementioned enzyme activation system and/or the balance between them and their inhibitors (tissue inhibitors of metalloproteinases), which bind to matrix metalloproteinases or their inactive pro-enzymes (Rabot et al., 2007). Stromal cell proliferation is mediated by the transforming growth factor-β1 (TGF-β1), a cytokine that acts through its receptors (TGF-βR1 and TGF-βR2) (Plaut et al., 2003) and activates fibroblasts to increase their capacity to synthetise proteins and proteases. The increase in mammary stromal area at the stage of active involution may result from increased production of stromal proteins, while the decrease at the stage of lactogenesis may be associated with the decreasing number of fibroblasts, which produce stromal proteins (De Vries et al., 2010). Moreover, IGFBP-5 interacts with proteins of the extracellular matrix (e.g., components of the plasminogen system or matrix metalloproteinases) involved in tissue remodeling during mammary involution (Flint et al., 2005). More precisely, IGFBP-5 enhances activity of tissuetype plasminogen activator, which activates plasminogen to plasmin. Then, plasmin initiates the degradation of the extracellular matrix, through activation of matrix metalloproteinases, resulting in tissue remodeling, which is correlated to involution. Hence, IGFBP-5 has a dual role; it coordinates apoptosis by decreasing availability of IGF-1 and promotes tissue remodeling by increasing extracellular matrix 4

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epithelial cells (Tatarczuch et al., 1997). During active involution, the alveolar area of the gland decreases, whilst its stromal area increases. Progressively, the amount of between-lobules connective tissue becomes abundant and dense (like a wide band). Increased numbers of fibroblasts are present therein. The stroma of connective tissue within the mammary lobules contains large numbers of inflammatory cells (primarily macrophages and lymphocytes) (Lee and Lascelles, 1969; Tatarczuch et al., 2000). The proportion of epithelial area in the mammary gland of ewes is minimal on the 8th day after cessation of lactation and on the 30th day of lactation (Colitti and Farinacci, 2009). It is noteworthy that its maximum proportion is reached on the 60th and the 150th day of a lactation period, at which time-points stromal area reaches its lowest (Colitti and Farinacci, 2009). The lumen area decreases subsequently to 8 days into involution. The cell proliferation index, which has its maximum during lactation (> 40% during lactation), is reduced to its lowest (< 10%) during involution. Rate of apoptosis is highest at the early stages of the process, but thereafter stabilises to a rate similar to that occurring at early lactation (Colitti and Farinacci, 2009). Initially, the outline of myoepithelial cells becomes irregular and distinct cytoplasmic processes enveloped by the basement membrane can be seen. Progressively, they become irregular in form and their cytoplasmic processes protrude deep into the glandular stroma. Finally, they decrease in size and their cytoplasm is condensed around the nucleus (Tatarczuch et al., 1997). During involution, numbers of leucocytes (initially neutrophils, subsequently lymphocytes and macrophages) in the mammary gland increase sharply (Lee and Lascelles, 1969; Tatarczuch et al., 2000). On the 15th day of involution, there is a marked presence of many, highlyvacuolated macrophages in the alveolar lumens and in the ductal system, which are termed ‘cells of Donné’ (Tatarczuch et al., 1997). As involution progresses, the stroma of connective tissue within the lobules contains large numbers of these cells. The leucocytes participate in the involution process, specifically in the removal of fat globules, casein micelles and cellular debris, which may lead to decreased efficiency of phagocytosis and intracellular killing of bacteria during the procedure (Sordillo and Nickerson, 1988).

5. Defences of the mammary gland during involution An important defence structure during the ‘dry-period’ is the keratin plug, which is formed at the teat orifice and seals the teat duct, thus preventing invasion of microorganisms into the mammary parenchyma. The plug is formed by continuous cornification of the teat duct epithelium, which is not being removed as milking or suckling has ceased. The plug plays a role of mechanical barrier and, moreover, inhibits growth of microorganisms due to the antibacterial properties of the fatty acids, which are its major constituents (Hogan et al., 1986, 1987; Capuco et al., 1992). In any case, the keratin plug takes some time to be established. Until then, milk accumulation into the mammary gland leads to increased pressure to the teat, which results to dilatation of the teat orifice and teat duct, thus facilitating potential invasion of pathogens. Characteristically, formation of the plug takes longer in animals with increased milk yield, i.e., in those with larger milk accumulation and wider teat dilatation, which are thus at higher risk of infection (Dingwell et al., 2004). Moreover, lack of regular milking or suckling, contributing to removal of invading microorganisms, and discontinuation of teat sanitation practices would lead to increased risk of infection during the stage of active involution. The dominant cells in ewes’ milk during lactation are the macrophages; polymorphonuclear leucocytes and lymphocytes are also present therein (Lee and Outteridge, 1981). As lactation period progresses, proportions of macrophages and neutrophils increase, possibly in order to establish an activated immune defence status as drying-off approaches (Cuccuru et al., 1997). As discussed above, there is a marked increase of leucocytes in the mammary gland during involution. During early involution neutrophils are the predominant leukocytes in mammary secretions, but after the first week their population declines and macrophages are again the dominant cells (Tatarczuch et al., 2000, 2002). During the stage of active involution, leucocytes in the mammary gland may have a decreased phagocytic ability, because they are heavily laden with ingested material (fat globules, casein micelles, cellular debris) (Sordillo and Nickerson, 1988; Tatarczuch et al., 2000, 2002). Ingestion of fat globules or casein micelles through internalisation of cell membrane results in cell rounding and loss of pseudopodia, which are necessary for trapping and engulfing bacteria, as neutrophils lack ability to regenerate plasma membrane (Paape et al., 2003). Moreover, there is a loss of lysosomes, which fuse to vacuoles containing fat globules or casein micelles instead of those containing bacteria (Paape et al., 2003). Progressively, number and efficiency of leucocytes increase, hence, at the state of ‘steady-state’ involution the mammary gland is well protected against potential invading pathogens. One should also refer to the sub-epithelial lymphoid nodules located at the border between the teat duct and the teat cistern (Mavrogianni et al., 2005; Fragkou et al., 2010). These structures regulate the early defence response of the mammary gland through lymphocytes; hence, as during involution there is an influx of lymphocytes into the mammary gland, one may postulate that their defence ability would increase at that period. Concentration of immunoglobulins is low during the stage of active involution, hence not supporting effective bacterial opsonisation and phagocytosis. Later, however, during the stage of ‘steady-state’ involution, that concentration increases. Thus, at that stage, opsonisation of invading bacteria increases leading to effective phagocytosis (Sordillo et al., 1987). Another defensive factor, the concentration of which in mammary secretion increases at the involuting mammary gland, is lactoferrin. Lactoferrin acts by sequestering iron, which is necessary for growth of Gram-negative bacteria, some of which are confirmed mammary pathogens (e.g., Escherichia coli) (Bishop et al., 1976; Nickerson, 1989; Oliver and Sordillo, 1989). Citrates compete with lactoferrin for iron binding, but, when iron is bound by citrates, it becomes available to bacteria. Immediately after cessation of lactation, concentration of

4.2. Ultrasonographic changes In general, anatomic structures in the mammary gland of ewes have a medium echogenicity to hyperechogenicity. Blood vessels and lactiferous ducts can be imaged therein. At the beginning of involution, the mammary parenchyma includes (a) the lobular structures (consisting of alveolar areas with reduced echogenicity), (b) the connective tissue (with increased echogenicity) and the ductal part of the gland and (c) the blood vessels therein (imaged as anechoic antra). As involution progresses in ewes, the mammary parenchyma is imaged to have homogeneity, with disappearance of the lobular picture and a progressive decrease of its grey-scale. Moreover, an overall reduced echogenicity can be recorded. In the mammary gland of ewes, structures with medium echogenicity could include fat, ductal and lobular epithelial tissues and loose, intralobular and periductal stromal fibrous tissue. In contrast, hyperechoic structures could include the compact interlobular stromal fibrous tissue, the mammary fasciae, the ligaments and the skin. Milk clots can be imaged into the gland cistern of ewes about a week after cessation of milking. Initially, these appear as small flakes that ‘flow’ into the gland cistern, later aggregating to form large clots (1–2 cm in diametre), which remain visible ultrasonographically up to one month after milk removal had stopped (Fig. 1). A temporary increase in cistern area is evident in the first week and is followed by a subsequent decrease (Fig. 2).

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Fig. 1. Serial ultrasonographic appearance of mammary parenchyma [mp] of ewes during progressive udder drying-off procedure (clockwise, from upper left photograph) (a) D0 (before start of the drying-off procedure), (b) D14, (c) D30, (d) D37. Images taken at a level after the branching of the external pudendal artery (when distance between the two branches [b] was ˜1 cm) on a MyLab® 30 ultrasonography system with linear transducer 10.0 MHz and scanning depth 60 mm (Petridis, 2014).

lactoferin in mammary secretion is minimal, which increases susceptibility of the mammary gland to infections. Subsequently, during the stage of ‘steady-state’ involution, concentration of lactoferrin is increased, whilst citrates have been resorbed, which further enhances the potential anti-bacterial action of lactoferrin (Smith and Oliver, 1981; Nickerson, 1989; Oliver and Sordillo, 1989). Thus, it becomes evident that the mammary gland is at high risk to infection and development of disease, termed ‘dry-ewe’ mastitis, during

the first two weeks after cessation of a lactation period (Barkema et al., 1998; Saratsis et al., 1998). In view of that and as part of routine health management of pregnant ewes, intramammary administration of antimicrobial agents at the end of a lactation period has been advocated; objectives of the procedure are (i) to cure infections which have occurred during the previous lactation and (ii) to prevent development of new mammary infections during the dry period (Contreras et al., 2007; Fthenakis et al., 2012). Subsequently, during the stage of ‘steady-state’

Fig. 2. Serial ultrasonographic appearance of gland cistern [gc] of ewes during abrupt udder drying-off procedure (from left to right): (a) D0 (before start of the drying-off procedure), (b) D7, (c) D30. Images taken at an inclined dorsal imaging plane, from the upper part of the intermammary groove towards the teat, which was used as the scanning axis on a MyLab® 30 ultrasonography system with microconvex transducer 3.3 MHz and scanning depth 120 mm (Petridis, 2014). 6

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involution, all the above-mentioned defence mechanisms are fully functional and lead to effective protection of the mammary gland. Finally, at the stage of redevelopment and lactogenesis, i.e. before the expected lambing, as the peri-parturient relaxation of immunity is established (Coop and Kyriazakis, 1999), numbers and phagocytic efficiency of leucocytes in the mammary gland decrease, as do amounts of lactoferrin and cytokines. Moreover, decreased cytokine production at that stage also leads to reduced chemotaxis of neutrophils to the mammary gland. Specifically, CD4+ cells produce small quantities of interleucin-2 and IFN-g, but larger ones of interleucin-4 and interleucin10 (Shafer-Weaver et al., 1999). That pattern has been correlated with increased susceptibility to mastitis, as decreased levels of those immunoregulatory cytokines had been correlated with impaired immune cell function and increased susceptibility to mastitis (Sordillo et al., 1991); for example, interleucin-2 activity has been found to be significantly smaller in mammary secretion samples collected immediately pre-partum than in samples collected 14 days prior to that (Sordillo et al., 1991). Although that way susceptibility of the mammary gland may increase, presence of keratin plug at the teat orifice minimises invasion of pathogens, whilst persistent efficacy of antibiotics that had been previously administered intramammarily can contribute to counteract potential infections (Hurley, 1989; Bradley and Green, 2004; Petridis et al., 2012; Petridis and Fthenakis, 2014).

flock.

• Decrease of flock bulk somatic cell counts in the subsequent lactation period.

It is also noteworthy that lambs (especially in large litters) from ewes with extensive mammary lesions do not thrive well and may require additional feeding (Fthenakis and Jones, 1990), which increases expenses and labour in the flock. The above procedures should be complemented by administration (preferably by the intramammary route) of antibiotics, which is an integral part of udder health management (Fthenakis et al., 2012; Petridis and Fthenakis, 2014). Since the mammary gland is more susceptible to new infections during the stage of early involution, as mentioned above, there is an effort among researchers to develop a strategy that would decrease milk production before drying off and would accelerate mammary involution. The use of substances, which inhibit release of prolactin, e.g., quinagolide and cabergoline, can reduce milk secretion at the end of a lactation period and hasten mammary involution, thus rendering the mammary gland less susceptible to potential mammary infections during the dry period. Thusfar, use of such substances has been studied only in cows. Quinagolide hastens the involution process (Ollier et al., 2013) without causing metabolic stress (negative energy balance), as is the case with feed restriction at the end of the lactation period as a method of decreasing milk production before drying off (Ollier et al., 2014), a practice that can lead to adverse welfare effects for animals subjected to that. Hastened mammary involution results in reduced susceptibility to intramammary infections (Ollier et al., 2015). Cabergoline decreases udder pressure and subsequent milk leakage at drying off, as well as signs of udder pain due to engorgement after cessation of lactation (Bach et al., 2015; Bertulat et al., 2017); it accelerates involution process by reducing secretory activity of mammary epithelial cells and enhances local immune defense (Boutinaud et al., 2016). Advantage of cabergoline over quinagolide is that it needs to be administered only once, while quinagolide should be administered several times for optimum efficacy. Although in cows results supportive of using these have been reported, the findings cannot be directly extrapolated in ewes, due to the physiological differences between the two species. Another approach for hastening involution process are the use of infusions of chitosan hydrogels. These can also activate immune response of mammary glands, that way contributing to reducing risk of new infections and disease during the dry period (Lanctôt et al., 2017). Their use is compatible with the simultaneous use of teat sealants (Lanctôt et al., 2017). The involution process can be affected by environmental factors, e.g., temperature or photoperiod. Heat stress during the dry period compromises mammary gland development before parturition by decreasing mammary cell proliferation and, thus, quantity of milk produced during the subsequent lactation period (Tao et al., 2011). Moreover, it affects negatively immune function during the transition period (do Amaral et al., 2010, 2011). Photoperiod during the dry period interferes also with involution of the mammary gland; specifically, during short days, there is increased mammary cell proliferation and decreased apoptosis, enhancing that way remodeling of the tissue (Wall et al., 2005), improving immune function (Auchtung et al., 2004) and milk production during the next lactation period (Miller et al., 2000; Auchtung et al., 2005).

6. Udder health management in ewes at the end of a lactation period 6.1. Approaches to efficient udder health management at the end of a lactation period Udder health management at the end of a lactation period aims to the following (Fthenakis et al., 2012).

• Cure of infections that have occurred during the previous lactation period. • Prevention of new intramammary infections during the ‘dry-period’. Cure of existing infections is significant for the ongoing involution process and the production of milk during the subsequent lactation period. It has been shown that long-standing intramammary infections with Staphylococcus aureus during the dry period can interfere with normal involution procedure, by favouring proliferation of parenchymal and stromal mammary cells (Andreotti et al., 2017), resulting in increase of the interlobular stromal area and permanence of nonsecretory parenchymal tissue (Andreotti et al., 2014). As a first step, clinical examination of the udder of ewes in the flock should be carried out, in order to identify ewes with mammary abnormalities. The udder of all ewes in the flock is examined by palpation, whilst the animals are run through a race (Orphanou, 1987; Saratsis et al., 1998). If mammary abnormalities are suspected, animals should be individually examined. Diffuse hardness, abscesses and nodules in the mammary glands are the most common clinical findings during the examination (Saratsis et al., 1998). Samples (e.g., mammary secretion, abscess material) should also be collected for bacteriological examination (Fthenakis, 1994; Saratsis et al., 1998; Mavrogianni et al., 2005). Based on results of clinical examination of the udder and of ancillary tests performed (e.g., bacteriological examination), the following categories of ewes should be considered for culling.

• Animals with at least one mammary gland permanently damaged. • Animals chronically affected. • Animals with incidents of relapsing mastitis or not fully respondent to mastitis treatment during the preceding lactation period. • Benefits of culling such animals are as follows. • Decrease of veterinary expenses for mastitis control in the flock. • Elimination of sources of potential infection for other animals in the

6.2. Appraisal of intramammary administration of antibiotics The efficacy of intramammary antibiotic administration at the end of a lactation period for the cure of existing mammary infections has been well documented (Petridis and Fthenakis, 2014). Cure rate after administration of antibiotics has been reported to range from 60% to 7

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Table 1 Categorisation* of antimicrobial agents based on the degree of risk to humans due to antimicrobial resistance development following use in animals (European Medicines Agency, 2019). Category

Risk to public health

Antimicrobials licence for animals

Advice on use

1 2

Low / Limited Higher

Narrow spectrum penicillins, macrolides, tetracyclines Fluoroquinolones, systemic 3rd / 4th generation cephalosporins, (aminoglycosides, broad-spectrum penicillins), colistin

General principles of responsible use to be applied Restricted to use where there are no alternatives or response to alternatives expected to be poor

* table valid at the time of publication.

82% (Petridis and Fthenakis, 2014). Use of antibiotics before the beginning of the dry period also contributes to reduced incidence of mastitis during the dry period (Linage and Gonzalo, 2008). The large variations in cure rates reported in the relevant studies can be explained by the variety of bacteria causing mastitis and the administration of antimicrobial agents active against only some of these organisms or against which causal bacteria have developed resistance. Self-cure of intramammary infections and mastitis also occurs during the dryperiod. Self-cure rate can vary from 6.5% to 60% (Paape et al., 2001; Contreras et al., 2007; Petridis and Fthenakis, 2014), which may depend upon the virulence of causal bacteria and the innate defence ability of the affected ewes. The practice can also result in decrease of bulk milk somatic counts in flocks during the subsequent lactation period (Gonzalo et al., 2009). The effects have been reported to be more pronounced in machinemilked flocks and have been associated with increased milk production and weight increase of lambs in mutton-production systems, as a consequence of the increased milk production of the ewes (Watson and Buswell, 1984; Fthenakis and Jones, 1990; Shwimmer et al., 2008). Those beneficial results of using antibiotic tubes are independent of procedure followed for udder drying-off (progressive or abrupt) (Petridis et al., 2012), of antibiotic administration approach followed (complete or selective) (Gonzalo et al., 2004) and of length of dry period (Linage and Gonzalo, 2008). The European Commission (2015) has now recommended “avoiding the systematic treatment of cows at drying-off, and considering and implementing alternative measures on a case-by-case basis”. Although the recommendation does not specifically include ewes, this may possibly be implied as the relevant section of the official document is titled as ‘6. Disease prevention and reducing the need to use antimicrobials – 6.4. Bovine and small ruminants’ (European Commission, 2015). Consequently and in order to decrease risk of potential antibiotic residues in the food chain and of increased incidence of antibiotic resistant bacterial strains in animals, selective administration should be performed. Further, this approach reduces costs and decreases risk of potential iatrogenic contamination of the mammary glands. However, its disadvantage is incomplete protection against new infections during the dry period, which may be overcome by using other tools for prevention of mastitis, e.g., vaccination (Petridis and Fthenakis, 2014; Fthenakis and Gonzalez-Valerio, 2017). During application of the selective approach for antibiotic administration, identification of animals may be based on combination of results of clinical examination of the udder of all animals and California Mastitis Test (CMT) (Politis et al., 2017). There is a strong correlation between CMT scores and milk somatic cell counts in sheep; CMT scores depend on somatic cell count, as well as on cell ratio changes, specifically on changes of proportions of neutrophils (Souza et al., 2012). If resources allow, the selection procedure could be optimised by performing bacteriological examination of samples from clinically affected udders, results of which would indicate selection of the most appropriate antibiotic for use (Orphanou, 1987; Saratsis et al., 1998; Petridis and Fthenakis, 2014). In all cases, the selection of the most appropriate antimicrobial agent is the responsibility of the attending veterinarians, who are the only ones licenced to prescribe antimicrobial agents for animals.

Moreover, in considering the antimicrobials to be used on each occasion, one should always take into account their categorisation, based on the degree of risk to humans due to antimicrobial resistance development following use in animals (Table 1) (European Medicines Agency, 2019). In all cases, the selection procedure of antimicrobial(s) to administered should be performed on the basis of the bacteria isolated in flocks, as well as on the results of susceptibility testing of organisms isolated (Vasileiou et al., 2019). The whole procedure should be performed under good hygienic conditions with disinfection of the teat preceding infusion of the antibiotic, in order to avoid iatrogenic contamination of the mammary gland. If possible, the tip of the antibiotic tube should be only partially inserted to avoid excessive dilatation of the teat canal and destruction of its lining (Bergonier et al., 2003; Gonzalo et al., 2004). After administration, definite and complete cessation of lactation is essential for success of the procedure and the prescribed withdrawal periods should be maintained to safeguard the public health (Fthenakis et al., 2012). 7. Concluding remarks Mammary involution is important for optimum function of the gland in the subsequent lactation period. At the same time, the mammary gland becomes susceptible to infections, which would affect normal involution process. Therefore, appropriate udder health management should take place to guarantee health of the mammary gland and subsequent optimum milk production. In sheep, mastitis has adverse financial consequences and has also been described as the most important problem affecting welfare of ewes. This paper has provided a detailed review of udder physiology and health management in dry-ewes, which is a significant task in sheep flocks within their annual production cycle. The procedures will need to be adapted in individual flocks after taking into account their specific requirements. Strategic udder health management and a mastitis prevention scheme in sheep flocks will minimise the incidence and the impact of the disease. References Akers, R.M., Capuco, A.V., Keys, J.E., 2006. Mammary histology and alveolar cell differentiation during late gestation and early lactation in mammary tissue of beef and dairy heifers. J. Drug Deliv. Sci. Technol. 105, 44–49. Andreotti, C.S., Pereyra, E.A.L., Baravalle, C., Renna, M.S., Ortega, H.H., Calvinho, L.F., Dallard, B.E., 2014. Staphylococcus aureus chronic intramammary infection modifies protein expression of transforming growth factor beta (TGF-b) subfamily components during active involution. Res. Vet. Sci. 96, 5–14. Andreotti, C.S., Pereyra, E.A.L., Sacco, S.C., Baravalle, C., Renna, M.S., Ortega, H.H., Calvinho, L.F., Dallard, B.E., 2017. Proliferation-apoptosis balance in Staphylococcus aureus chronically infected bovine mammary glands during involution. J. Dairy Res. 84, 181–189. Antonsson, B., 2004. Mitochondria and the Bcl-2 family proteins in apoptosis signaling pathways. J. Supramol. Struct. Cell. Biochem. Suppl. 256–257, 141–155. Auchtung, T.L., Rius, A.G., Kendall, P.E., McFadden, T.B., Dahl, G.E., 2005. Effects of photoperiod during the dry period on prolactin, prolactin receptor, and milk production of dairy cows. J. Dairy Sci. 88, 121–127. Auchtung, T.L., Salak-Johnson, J.L., Morin, D.E., Mallard, C.C., Dahl, G.E., 2004. Effects of photoperiod during the dry period on cellular immune function of dairy cows. J. Dairy Sci. 87, 3683–3689. Bach, A., De-Prado, A., Aris, A., 2015. Short communication: the effects of cabergoline administration at dry-off of lactating cows on udder engorgement, milk leakages, and lying behavior. J. Dairy Sci. 98, 7097–7101.

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