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Small Ruminant Research 152 (2017) 86–99

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Ultrasonographic examination of the udder in sheep M.S. Barbagianni, V.S. Mavrogianni, N.G.C. Vasileiou, G.C. Fthenakis, I.G. Petridis ∗ Veterinary Faculty, University of Thessaly, 43100, Karditsa, Greece

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Article history: Available online 21 December 2016 Keywords: Cistern Diagnosis Ewe Mammary gland Mammary parenchyma Mastitis Milk yield Sheep Teat Ultrasonography

a b s t r a c t Objective of the paper is to review work relevant to ultrasonographic examination of the udder of ewes. Udder structures that can be readily imaged ultrasonographically (B-mode or Doppler examination) are mammary parenchyma, the gland cistern, the lactiferous ducts, the mammary vessels, the teat and the supramammary lymph nodes. Conventional diagnostic approaches for mastitis (i.e., combination of clinical, bacteriological and cytological examinations) provide a good means for diagnosis of mastitis; hence, in diagnosis of mastitis, use of ultrasonographic examination has, in general, an ancillary role, for example during investigation of cases, in which clinical diagnosis alone can prove of little help, e.g., in animals with small-sized, deep mammary nodules. The technique can be used at the end of a lactation period as part of routine udder examination performed at that point, which would provide additional useful information, e.g., regarding presence of abcesses or of increased quantity of fibrous tissue. Further, estimation of the dimensions of gland cistern of ewes in a flock would support decisions regarding milking frequency to be applied. Finally, examination of the teat can provide indications regarding optimising use of the milking machine by applying the appropriate settings. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The importance of the mammary glands in the profitability of ewes, especially in dairy flocks, makes early and accurate diagnosis of its disorders necessary. This will, in turn, support efficient management of cases of diseased animals. The matter is of significance for the welfare of the affected animals (European Food Safety Authority, 2014), as well for their productivity in terms of quantity and quality of milk produced. The diagnosis of clinical or subclinical mastitis in ewes has been recently reviewed by Fragkou et al. (2014). Various approaches can be employed, which more often include clinical, bacteriological and/or cytological techniques and methodologies. Use of ultrasonography has also been advocated and various publications have indicated the usefulness of the technique in the diagnosis of mammary diseases of ewes. Further, the technique can be employed in management practices in healthy animals, e.g., for selection of animal for increased milk production, based on the volume of the gland cistern. Finally, other workers have used the methodology for research purposes to support the study of various mechanisms in healthy or diseased animals. Objective of the current paper is to

∗ Corresponding author. E-mail address: [email protected] (I.G. Petridis). http://dx.doi.org/10.1016/j.smallrumres.2016.12.009 0921-4488/© 2016 Elsevier B.V. All rights reserved.

review work relevant to ultrasonographic examination of the udder of ewes. 2. Methodology of ultrasonographic examination 2.1. Principles of ultrasonographic examination of the udder of ewes Ultrasonographic examination can be used to explore the structure of the udder of ewes, as well as to study functional parametres of the organ (Ruberte et al., 1994; Caja et al., 1999; Nudda et al., 2000; Petridis et al., 2014; Barbagianni et al., 2015). By using ultrasonographic examination, monitoring of the udder can be performed and information may be obtained regarding its internal structures, as ultrasonographic images of the mammary gland correspond well to its anatomical structure (Ruberte et al., 1994). The technique can be used for examination of the mammary parenchyma, the gland cistern and/or the teat (Franz et al., 2001) and the supramammary lymph nodes (Hussein et al., 2015), as well as for evaluation of blood flow disorders (Petridis et al., 2014; Barbagianni et al., 2015; Barbagianni, 2016). Further, during ultrasonographic examination details are collected in real time, as processes are developing. Udder structures that can be readily imaged ultrasonographically are the body of the mammary gland (corpus mammae) with the mammary parenchyma, the gland cistern (sinus lactiferous),

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Fig. 1. B-mode ultrasonographic presentation of mammary parenchyma; image taken at the 6th month of lactation period, along the long axis of the udder; mildly echogenic ® mammary parenchyma and section of the external pudendal artery with its echogenic wall and its three branches. Image taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with microconvex transducer, imaging frequency: 3.3 MHz – scanning depth: 120 mm.

Table 1 Summary of references regarding use of transducers during ultrasonographic examination of various structures of the udder of ewes. Reference

Details of transducer employed

Part of udder examined

Ruberte et al. (1994) Nudda et al. (2000) Franz et al. (2001) Franz et al. (2003) Mavrogianni et al. (2004) Wójtowski et al. (2006) Castillo et al. (2008) Rovai et al. (2008) Alejandro et al. (2014b) Petridis et al. (2014)

5.0 MHz sector 3.5 MHz convex sector 12.0 MHz linear 8.5 MHz 6.0 MHz sector 10.0 MHz linear 5.0 MHz sector 5.0 MHz sector 5.0 and 7.5 MHz linear 10.0 MHz linear 3.3 MHz microconvex 10.0 MHz linear 7.5 MHz linear and 4.0 MHz convex 12.0 MHz linear

Gland cistern Gland cistern Teat (examination through water filled plastic cup) Teat, gland cistern, mammary parenchyma Teat (direct application) Teat (examination through water filled plastic cup) Gland cistern Gland cistern Teat (examination through water filled plastic cup) Mammary parenchyma Gland cistern

Barbagianni et al. (2015) Hussein et al. (2015) Barbagianni (2016)

the lactiferous ducts (ducti lactiferi), the mammary vessels [especially the external pudendal artery (arteria pudenda externa) with the three branches: caudal mammary artery (arteria mammaria caudalis), mid mammary artery (arteria mammaria media), cranial mammary artery (arteria mammaria cranialis) (Fig. 1), as well as the larger mammary veins: mid mammary vein (vena mammaria media), cranial mammary vein (vena mammaria cranialis), external pudendal vein (vena pudenda externa)], the teat (papilla mammae) with the teat duct (ductus papillaris), the teat cistern (sinus papillaris) and the teat arteries (arteriae papillares), and the supramammary lymph nodes (lymphonodi inguinalis superficialis). Benefits from using udder ultrasonographic examination depend strongly on the operator’s experience; more experienced operators would produce better images, with increased repeatability, and leading to more accurate interpretation (Klein et al., 2005; Díaz et al, 2013). The ultrasonographic examination of the mammary glands in sheep can be performed with the animal in the standing position, under mild restraint by an assistant. Wherever there is availability, the examination can take place in the milking parlour, which

Mammary parenchyma Teat, mammary parenchyma, supramammary lymph nodes Teat (direct application)

would improve work-flow and ease of work for the operator and also decrease time required for the examination. Hairs on the udder should be clipped, to facilitate the procedure and to obtain improved image. Coupling gel should be applied on the udder and the examination starts by placing the transducer on the caudal surface of the udder. The type of transducer employed for the examination differs depending on the available equipment, the structure of the udder and the specific structure under examination. Types of transducers referenced in the literature are summarised in Table 1. 2.2. Ultrasonographic examination of mammary parenchyma For ultrasonographic examination of the parenchyma, a linear transducer is used most often. This should be placed transcutaneously, in a position perpendicular to the long axis of the udder. Initially, dorsal sections of the mammary parenchyma are taken, starting from the upper part downwards; then, the transducer is moved around the axis of the udder. The images can be evaluated immediately or can be saved for further processing and detailed

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Fig. 2. B-mode ultrasonographic presentation of gland cistern; image taken at the 6th month of lactation period, in an inclined sagittal scan towards the teat; presence of ® small clots (arrow) floating inside the cistern. Images taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with microconvex transducer, imaging frequency: 3.3 MHz – scanning depth: 120 mm.

Fig. 3. B-mode ultrasonographic presentation of the gland cistern; image taken at the 6th month of lactation period, in an inclined sagittal scan towards the teat; presence of a ® large clot (arrow) floating inside the cistern. Images taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with microconvex transducer, imaging frequency: 3.3 MHz – scanning depth: 120 mm.

evaluation later. Scanning depth and frequency employed vary and depend on the size of the udder examined, as well as on the available equipment. A frequency of 7.5–10.0 MHz can be used in most cases (Petridis et al., 2014; Barbagianni et al., 2015); a smaller frequency may be occasionally needed in animals with a larger udder. One mammary gland is imaged initially, which is followed by imaging of the contralateral gland. There is particular scope for analysis of grey-scale intensity of images of mammary parenchyma (Petridis et al., 2014; Barbagianni et al., 2015). This would provide indirect information regarding amount of fluids (i.e., lacteal secretions) into the parenchyma and

also, possibly, about the amount of glandular tissue. In an image processing context, grey-scale analysis refers to the image’s overall pixel grey intensity values (Ojala et al., 2002) and can be performed by means of various image processing softwares (e.g., ImageJ; National Institutes of Health, Rockville Pike, MD, USA). For analysis of grey-scale of the whole parenchyma, it is recommended to evaluate intensity values of at least three images of the parenchyma, each presenting a different part of the parenchyma with the median value calculated at the end; areas with vessels or ductal formations should not be taken into account for the grey-scale analysis (Petridis et al., 2014; Barbagianni et al., 2015). In analysis of grey-scale inten-

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Fig. 4. B-mode ultrasonographic presentation of mammary parenchyma; image taken on the penultimate week of pregnancy, at a level immediately above the gland cistern; non-homogeneous mammary parenchyma, due to incomplete development during lactogenesis (for fully developed mammary parenchyma: Fig. 5). Image taken ® and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 10.0 MHz – scanning depth: 60 mm.

Fig. 5. B-mode ultrasonographic presentation of mammary parenchyma; image taken on the last week of pregnancy, at a level immediately above the gland cistern; homogeneous, mildly echogenic fully developed mammary parenchyma, with no abnormalities evident therein (for underdeveloped mammary parenchyma: Fig. 4). ® Image taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 10.0 MHz – scanning depth: 60 mm (Barbagianni et al., 2015).

sity of images, results are expressed on a 0 (black) to 255 (white) scale. 2.3. Ultrasonographic examination of gland cistern After examination of the parenchyma, the gland cistern can be examined. The gland cistern can be better visualised by means of a microconvex or sector transducer. An inclined sagittal imaging plane should be taken, starting from the upper part of the intermammary groove (sulcus intermammarius) towards the teat, which should be used as the scanning axis. That way, the size of the cistern can be initially estimated; then, by moving the transducer to the left and the right, upwards and downwards, after a 90◦ rotation, it is possible to check for clots, fibrous tissue development/formation and/or lactoliths (Figs. 2, 3). By means of the inclined sagittal image, the whole cistern can be visualised in one scan (Ruberte et al., 1994). Nudda et al. (2000) have proposed the application of the transducer on the inguinal-abdominal fold at the side of the udder and, by hav-

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Fig. 6. B-mode ultrasonographic presentation of mammary parenchyma; image taken on the last week of pregnancy, at a level immediately above the gland cistern; homogeneous, mildly echogenic fully developed mammary parenchyma, with pres® ence of well-developed lactiferous ducts. Image taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 10.0 MHz – scanning depth: 60 mm.

Fig. 7. B-mode ultrasonographic presentation of mammary parenchyma; image taken on the last week of pregnancy at the level of the gland cistern; presence of well-developed lactiferous ducts and gland cistern. Image taken and processed ® on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 10.0 MHz − scanning depth: 60 mm.

ing the teat as a scan axis, to take cross sections of the contralateral gland cistern. Bruckmaier and Blum (1992) have created verticallycut images with the scanning plane longitudinally through the teat canal; cross sections of the entire udder, including the parenchyma and teat cisternal cavities, were examined vertically through the teat from below. However, the approach would be time-consuming and difficult to perform in field conditions (Bruckmaier and Blum, 1992; Bruckmaier et al., 1997). In animals with large cisterns, a decreased frequency, e.g. 3.5 MHz, would be required and an adequate scanning depth should be selected, in order for the entire cistern to be visualised (Castillo et al., 2008; Rovai et al., 2008). Measurement of the volume of the cistern can be difficult, because of the irregular shape of the structure; hence, only measurements of the area of one or more cross sections can be taken (Nudda et al., 2000); nevertheless, there is scope for use of three-dimensional ultrasonography, which might possibly improve direct evaluation of the volume of gland cistern. In any case, if such an examination

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Fig. 8. B-mode ultrasonographic presentation of mammary parenchyma; image taken on the 3rd week of lactation period at the level of the branching of the external pudendal artery; homogeneous, mildly echogenic mammary parenchyma, with no abnormalities evident therein; the external pudendal artery and the external puden® dal vein are imaged vertically sectioned. Image taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 10.0 MHz – scanning depth: 60 mm.

Fig. 9. B-mode ultrasonographic presentation of mammary parenchyma; image taken on the 3rd week of lactation period at the level of the branching of the external pudendal artery; homogeneous, mildly echogenic mammary parenchyma, with no abnormalities evident therein; vertical sections of the external pudendal artery and ® its branches are imaged. Image taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer imaging frequency: 10.0 MHz – scanning depth: 60 mm.

would be undertaken, it has been indicated that an interval of 8 h between milking and imaging should be maintained, as this leads to improved results in analysis of the relevant data (Castillo et al., 2008). 2.4. Ultrasonographic examination of teat The teat can be immersed into a plastic cup filled with warm water, to avoid its deformation during imaging and visualisation (Franz et al., 2001), with the transducer placed outside the cup. Alternatively, the teat could be filled with milk and the transducer

Fig. 10. B-mode ultrasonographic presentation of mammary parenchyma; image taken on the 3rd week of lactation period at the level immediately after the branching of the external pudendal artery; homogeneous, mildly echogenic mammary parenchyma, with no abnormalities evident therein. Image taken and processed ® on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 10.0 MHz – scanning depth: 60 mm.

Fig. 11. B-mode ultrasonographic presentation of mammary parenchyma; image taken on the 2nd month of lactation period at the level immediately after the branching of the external pudendal artery; homogeneous, sub-echogenic mammary parenchyma, with no abnormalities evident therein. Image taken and processed ® on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 10.0 MHz – scanning depth: 60 mm (Petridis et al., 2014).

placed directly on the teat skin after gel has been applied on it (Mavrogianni et al., 2004; Barbagianni, 2016). Franz et al. (2001) have indicated that a high frequency of 12.0 MHz is necessary for good imaging of the teat structures, although Díaz et al. (2013) have reported that, for imaging of the layers of the teat wall, a 7.5 MHz transducer would be more useful. Scanning starts from the base of the teat and continues towards its orifice (Mavrogianni et al., 2004; Fragkou et al., 2014). Scanning depths of 20–30 mm can be used during the examination. 2.5. Ultrasonographic examination of supramammary lymph nodes For examination of the supramammary lymph nodes, the transducer is placed on the area dorsal and lateral to the caudal aspect of udder halves. The lymph nodes can be easily identified from

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Fig. 12. B-mode ultrasonographic presentation of mammary parenchyma; image taken on the 2nd month of mammary involution at the level immediately after the branching of the external pudendal artery; homogeneous, significantly sub-echogenic mammary parenchyma, with no abnormalities evident therein. Image taken and processed on a ® MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 10.0 MHz – scanning depth: 60 mm (Petridis et al., 2014).

Fig. 13. B-mode ultrasonographic presentation of mammary parenchyma; image taken 3 days after development of mastitis associated with Mannheimia haemolytica, at the level immediately before branching of the external pudendal artery; nonhomogeneous mammary parenchyma with areas of increased echogenicity. Image ® taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 10.0 MHz – scanning depth: 60 mm.

their well-demarcated echogenic capsule and their echogenic linear structure that runs longitudinally through their centre (Kofler et al., 1998; Hussein et al., 2015). The two lymph nodes can be easily distinguished between them, if the ipsilateral branch of the mammary artery is followed. 2.6. Ultrasonographic examination of udder blood vessels The mammary blood vessels are best imaged by using Doppler ultrasonographic examination. In Doppler examination, blood vessels would appear blue or red, in accord to the movement of the red cells within the vessels (moving towards or away from the probe)

Fig. 14. B-mode ultrasonographic presentation of mammary parenchyma; image taken 3 days after development of mastitis associated with Mannheimia haemolytica, at the level immediately before the gland cistern; non-homogeneous mammary parenchyma with areas of increased echogenicity and foci of early stage of devel® opment of fibrous tissue (arrows). Image taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 10.0 MHz – scanning depth: 60 mm.

and the settings implemented in the ultrasound machine. That way, the external pudendal artery and its branches can be easily identified. Initially, the transducer is placed perpendicular to the long axis of the gland near its attachment to the abdomen; once the external pudendal artery has been localised, the transducer is rotated 90◦ and moved gently to the left and the right, in order to get a longitudinal/lengthwise cross-section of the vessel and its branches (Fig. 1). Then, the colour Doppler gate is positioned within the vessel examined, with no contact to the wall; when optimal flow is achieved, a spectral mode waveform is taken. Uniform waveforms from three consecutive cardiac cycles of the animal under examination should be considered for calculations (Petridis et al., 2014; Barbagianni et al., 2015). The technique can also be applied in the subcutaneous vessels of the teat. In Doppler mode examination

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Fig. 15. B-mode ultrasonographic presentation of mammary parenchyma; image taken 1 day after development of mastitis associated with Mannheimia haemolytica; normal ultrasonographic appearance lost in the imaged fields; non-homogeneous mammary parenchyma with areas of increased or reduced echogenicity indicative of the extensive parenchymal damage; presence of hypoechoic oedematous foci (red arrows), as well as hyperechogenic areas and foci of early stage of development of fibrous tissue around the external pudendal artery (yellow arrow). Image taken ® and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 10.0 MHz – scanning depth: 60 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 17. B-mode ultrasonographic presentation of mammary parenchyma, into which a hard formation was palpated at clinical examination; presence of nodulartype formation full with hypoechogenic material; arrows indicate artefact caused by the difference in echogenicity between the content of the formation (liquid) and the neighbouring parenchyma (when sound waves pass through fluid-filled formations, decrease of reflectance occurs, leading to acoustic enhancement thereafter). Image ® taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 10.0 MHz – scanning depth: 60 mm.

Fig. 18. B-mode ultrasonographic presentation of mammary parenchyma, into which a hard formation was palpated at clinical examination; presence of nodular-type formation full with hypoechogenic material, also with areas of hyperechogenicity; likely a mammary abscess (same artefact as in Fig. 17). Image taken ® and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 10.0 MHz – scanning depth: 60 mm.

Fig. 16. B-mode ultrasonographic presentation of mammary parenchyma, into which a hard formation was palpated at clinical examination; presence of nodulartype formation full with hypoechogenic material, surrounded by a hyperechogenic area; image intentionally cropped to avoid presentation of an artefact, caused by the difference in echogenicity between the content of the formation and the neighbour® ing parenchyma (for full image: Fig. 17). Image taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 10.0 MHz – scanning depth: 60 mm.

of the udder blood vessels, scanning frequency can vary from 5.0 to 6.6 MHz (Barbagianni, 2016). Further details regarding Doppler ultrasonography are available in another paper of this special issue (Petridis et al., 2017).

Fig. 19. B-mode ultrasonographic presentation of mammary parenchyma, into which hard formations were palpated at clinical examination; presence of two for® mations full with hypoechogenic material. Image taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 10.0 MHz – scanning depth: 60 mm.

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Fig. 20. B-mode ultrasonographic presentation of gland cistern; image taken on the 2nd month of lactation period at an inclined sagittal imaging plane, from the upper part ® of the intermammary groove towards the teat, used as the scanning axis; gland cistern at full size (for cistern at reduced size: Fig. 21). Image taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with microconvex transducer, imaging frequency: 3.3 MHz – scanning depth: 120 mm (Petridis et al., 2014).

Fig. 21. B-mode ultrasonographic presentation of gland cistern; image taken on the 2nd month of mammary involution at an inclined sagittal imaging plane, from the upper part of the intermammary groove towards the teat, used as the scanning axis; gland cistern at reduced size (for cistern at full size: Fig. 20). Image taken and processed on a ® MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with microconvex transducer, imaging frequency: 3.3 MHz – scanning depth: 120 mm (Petridis et al., 2014).

3. Findings during ultrasonographic examination of the udder 3.1. Findings in the mammary parenchyma The normal mammary parenchyma is imaged as a homogeneous, granular structure with medium echogenicity; anechoic structures identified therein correspond to lactiferous ducts and vessels. The image is the result of even distribution of more echoic connective tissue and less echoic mammary epithelial tissue; it also depends on the filling degree of the gland, as anechoic material (milk and lacteal secretions in general) would reduce the overall echogenicity of the image (Floeck and Winter, 2006; Franz et al., 2009; Barbagianni et al., 2015). Differences in the appear-

ance of the parenchyma according to reproductive status of the animals, i.e., pregnancy or after cessation of lactation, have been documented, which reflected changes occurring during lactogenesis or mammary involution, respectively (Figs. 4–12) (Petridis et al., 2014; Barbagianni et al., 2015). Various systemic disorders can have an effect in the echogenicity of the parenchyma; Barbagianni et al. (2015) have described that grey-scale intensity of mammary parenchyma in ewes with pregnancy toxaemia was increased in comparison with that in healthy individuals, but the echotexture of the image had not been altered. Ultrasonographic images of udders with mastitis reveal nonhomogenous regions in the mammary gland, in which hyper- and hypo-echogenic areas alternate (Franz et al., 2003), although, in some animals, echogenicity of the parenchyma may appear nor-

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Fig. 22. B-mode ultrasonographic presentation of teat; the teat cistern, which is full of milk, is imaged as anechoic cavity, with the teat duct marginally obvious at the right apex of the cavity, as a hyperechoic line; the hypoechoic parallel bands present ® in the teat wall, represent blood vessels. Image taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 12.0 MHz – scanning depth: 30 mm.

Fig. 24. B-mode ultrasonographic presentation of teat of a ewe with mastitis; the wall of the teat is imaged with increased echogenicity, with presence of particularly hyperechoic foci within the tissue; some flakes are also visible within the content of ® the teat cistern. Image taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 12.0 MHz – scanning depth: 30 mm.

Fig. 23. Colour Doppler ultrasonographic presentation of teat; the teat cistern, which is full of milk, is imaged as anechoic cavity, with the teat duct marginally obvious at the right apex of the cavity; presence of very few vessels in the subcuta® neous tissue. Image taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 6.6 MHz – scanning depth: 20 mm.

Fig. 25. Colour Doppler ultrasonographic presentation of teat; the teat cistern, which is full of milk, is imaged as anechoic cavity; presence of markedly increased blood flow into the teat vessels; image compatible with early stage of bacterial infection through the teat duct; teat duct imaged as an hyperechoic line with borders clearly defined as hypoechoic parallel bands. Image taken and processed on a ® MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 6.6 MHz – scanning depth: 30 mm.

mal, especially if only mild inflammation has developed. Further, milk can often be imaged into the gland cistern with the characteristic form of flakes; changes in the echogenicity of milk can also be evident, as milk becomes more echogenic and has the form of snow (Franz et al., 2009). In cases of severe mastitis, the normal ultrasonographic appearance is lost; the mammary parenchyma appears non-homogeneous with areas of increased or reduced echogenicity indicative of the extensive parenchymal damage; presence of hypoechoic oedematous foci, as well as of hyperechogenic areas may also be evident (Figs. 13–15). During infections with gas-forming bacteria, hyperechoic spots or bands casting dirty shadows in a homogeneous parenchyma could be found; in infections caused by Trueperella pyogenes, presence of hypoechoic spots with a hyperechogenic centre has been reported (Floeck and Winter, 2006; Franz et al., 2009). In cases of mastitis, mammary secretion, normally anechoic, can appear with increased echogenicity, as the result of increased cellular content therein (Floeck and Winter, 2006). Further, development of connective tissue, a feature of long-standing mastitis, might result in increase of

the echogenicity of the mammary parenchyma (Trostle and O’Brien, 1998). Ultrasonographic examination may also support diagnosis of mammary abscesses, when these have small size and are located deeply, thus not readily palpable (Hiepler et al., 2009) (Figs. 16–18). The technique can also help in the differentiation of mammary abscesses from haematomas or cysts, in which the image taken indicates thin hyperechoic septae or large clots floating in anechoic fluid. In cases of oedema located subcutaneously in the udder, apparent fluid-filled spaces are more extended than similar structures in haematomas; the former are also imaged as ‘the skin of the onion’, because of the alternating hyperechoic connective tissue and hypoechoic fluid (Floeck and Winter, 2006; Franz et al., 2009). In contrast, mammary abscesses are imaged as round structures with capsule and hypoechoic content (Floeck and Winter, 2006; Fasulkov, 2012). Depending on the stage of abscess formation, a hypoechoic capsule with hyperechoic content can also be found. Ultrasonographic evidence of gas shadowing within an encapsulated mass should be taken as supportive of the diagnosis of an

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Fig. 26. Colour Doppler ultrasonographic presentation of teat; the content of the teat cistern is imaged with increased echogenicity and disorderly appearance, with presence ® of hyperechoic foci; teat wall with increased echogenicity; development of neovascularisation in teat wall. Image taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, Doppler imaging frequency: 6.6 MHz – scanning depth: 20 mm.

abscess, although mixed echogenicity images can be obtained with both abscesses and haematomas (George et al., 2008) (Fig. 19). 3.2. Findings in the gland cistern The size of the gland cistern has been found to correlate well with milk production, hence ultrasonographic evaluation of its size has been proposed for the identification of animals with increased milk yield potential (Figs. 20, 21). That way, the technique may be used for selection of animals for improving milk production and milkability of ewes in a flock (Nudda et al., 2000; Castillo et al., 2008; Ayadi et al., 2011). Rovai et al. (2008) have concluded that selection of ewes for increased milk yield has resulted ultimately in breeding of animals with large gland cisterns (as confirmed by results of ultrasonographic examination), which indicates that greater volumes of milk can be accommodated therein. These animals can be more tolerant to long intervals between milkings (Rovai et al., 2008). Therefore, estimation of the size of the gland cistern can be used in choosing appropriate intervals between milkings, which would improve overall productivity in a flock (Labussiere, 1988; Nudda et al., 2000; Castillo et al., 2008). 3.3. Findings in the teat The teat wall has a three layer construction; below the external hyperechoic skin, there are the less echoic muscle and connective tissue layer and the hyperechoic mucosa layer. The teat cistern appears as a uniformly anechoic structure, better visualised when filled with milk (Franz et al., 2001); hypoechoic parallel bands, representing blood vessels may also be imaged therein (Figs. 22–24). During ultrasonographic examination of teats removed from dead ewes, the teat duct was imaged as a hyperechoic line around 8.0–9.0 mm in length and 0.2 mm in width, with borders clearly defined as hypoechoic parallel bands (Franz et al., 2001). The hyperechogenicity of the teat duct has been attributed to the stratified keratinised squamous epithelium (Franz et al., 2001). In cases of mastitis, the content of the teat cistern often contains hyperechogenic foci (Fig. 24). Increased vascularisation in the teat wall may become evident during Doppler examination (Figs. 25, 26). Ultrasonographic examination may also be useful in identifying the cause of teat stenosis (e.g., injury, lactoliths, polyps, papillomas, foreign body, congenital disorder) and establishing a prognosis in ewes with ensuing milk flow disorders. It may also support diagnostic differentiation between suspected conjoined teats

Fig. 27. B-mode ultrasonographic presentation of a teat, into which a hard, cord-like structure had been palpated lengthwise; presence of a long hyperechoic line under the mucosa of the teat cistern (S: skin; T: subcutaneous teat tissues; M: mucosa of the teat cistern; TC: teat cistern). Image taken and processed on an AMI B7 ultrasonography system (Alliance Medical, Quebec, Canada) with a sector transducer, imaging frequency: 6.0 MHz – scanning depth: 40 mm (Mavrogianni et al., 2004).

from teat fistulas (Franz et al., 2009). Imaging can reveal presence of haematomas or abscesses into the teat, as well as development of fibrous tissue within the teat, leading in stenosis. In experimentally induced teat stenosis, a hyperechoic line running lengthwise under the mucosa of the teat cistern has been observed; it has been hypothesised that this was the result of extensive fibrosis (Fig. 27) (Mavrogianni et al., 2004). Less often, foreign bodies may be observed into a teat, whilst the post-operative process after teat surgery may also be evaluated by ultrasonographic observation (Franz et al., 2009). Ultrasonographic examination has been used to evaluate effects of inappropriate milking technique in the health of teats (Alejandro

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Fig. 29. B-mode ultrasonographic presentation of mammary lymph node; image taken 25 days after development of mastitis associated with Staphylococcus chromogenes in the contralateral side of the udder; homogeneous parenchyma, with no abnormal features (when compared with contralateral lymph node: Fig. 28). Image ® taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 12.0 MHz – scanning depth: 40 mm.

Fig. 28. B-mode ultrasonographic presentation of mammary lymph node; image taken 25 days after development of mastitis associated with Staphylococcus chromogenes; marked enlargement of the lymph node, with thickening of the cortex ® (arrows). Image taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 12.0 MHz – scanning depth: 40 mm.

et al., 2014a), as changes in dimensions of the teat, as well as in its condition, might adversely affect with local defence mechanisms, leading in increased likelihood of infections (Mavrogianni et al., 2005, 2006). Overmilking may lead in increased teat wall thickness, due to congestion and oedema, which ultrasonographically is imaged as increase of the line perpendicular to the mid axis of the teat wall, when comparing findings before and after milking (Alejandro et al., 2014b). Inappropriate milking conditions can lead in significant increase in teat dimensions, most markedly 4 h after milking, which would not return to normal even 6 h later (i.e., 10 h after milking) (Wójtowski et al., 2006). Hence, ultrasonographic examination of teats can be employed to optimise milking conditions in dairy flocks, especially after installation of a new milking system. In a field investigation, Franz et al. (2003) have indicated a positive association between increased California Mastitis Test scores (reflecting cell content in a milk sample; Fthenakis, 1995) and increased diametre of teat canal (as measured during ultrasonographic examination). Therefore, ultrasonographic examination of the teat might be used to identify and select dairy ewes with long and narrow teat canals, which have been considered as factors minimising chances of mammary infection (Gelasakis et al., 2015), that way improving udder health in a flock. 3.4. Findings in the supramammary lymph nodes The supramammary lymph nodes have an anechoic or hypoechoic parenchyma, with the hilar area imaged as a highly echogenic linear structure. Their dimensions can vary, in accord with the health status of the ipsilateral mammary gland; in cases of mammary infection, their dimensions can be significantly increased (Figs. 28 and 29). According to Hussein et al. (2015), ultrasonographic examination of supramammary lymph nodes might possibly support diagnosis of subclinical mastitis in field conditions. It has been postulated that in ewes with subclinical mastitis, the supramammary lymph nodes had increased dimensions during ultrasonographic examination, leading to a conclusion that ewes in which length of these lymph nodes was ≥11.5 mm and width ≥7.8 mm, were 6.5

Fig. 30. B-mode ultrasonographic presentation of mammary parenchyma, showing the external pudendal artery and the external pudendal vein vertically sectioned; the larger section, appearing with more echogenic wall, represents the artery, whilst the smaller section, with less echogenic wall, represents the vein. Image taken and ® processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, imaging frequency: 12.0 MHz – scanning depth 40 mm.

times more likely to have subclinical mastitis (Hussein et al., 2015). Further, it has also indicated that in cases of staphylococcal infections, the architecture of lymph nodes might change, with a loss of the normally highly echogenic hilus central area (Hussein et al., 2015). 3.5. Findings in the udder blood vessels The mammary vessels can be imaged as anechoic structures inside the mammary parenchyma (Fig. 30). Doppler examination helps to differentiate them from lactiferous ducts. There is little work with Doppler examination in mammary vessels of ewes. Blood flow into the mammary gland through the external pudendal artery has been found to decrease progressively during involution, with the speed of the decrease depending on the procedure followed for drying-off (i.e., progressive or abrupt cessation of lactation period) (Figs. 31, 32) (Petridis et al., 2014). Blood flow into the mammary gland increases progressively with lactogenesis at the last stage of pregnancy (Figs. 33–35) (Barbagianni et al., 2015). Further, blood flow into mammary glands of ewes with pregnancy toxaemia has been found to be greatly reduced (Barbagianni et al., 2015), as well as blood flow into mammary glands of lactating ewes that had developed pregnancy toxaemia at the preceding pregnancy (Barbagianni, 2016). Possibly, an association of blood

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Fig. 31. Spectral waveforms of the external pudendal artery (Doppler ultrasonography) obtained 2 days after start of the drying-off procedure; increased blood input into ® the, still wide, vessel (for comparison with image obtained later during the involution process: Fig. 32). Image taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, Doppler imaging frequency: 6.6 MHz – scanning depth: 60 mm (Petridis et al., 2014).

Fig. 32. Spectral waveforms of the external pudendal artery (Doppler ultrasonography) obtained on the 5th week after start of the drying-off procedure; markedly reduced ® blood input into the vessel, which has a small diametre (for comparison with image obtained at start of involution process: Fig. 31). Image taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, Doppler imaging frequency: 6.6 MHz – scanning depth: 60 mm (Petridis et al., 2014).

input into the udder during late gestation with milk production during the subsequent lactation period could support a method to identify in advance ewes with increased milk yield in the forthcoming lactation period (Barbagianni et al., 2015). After infection, the diametre of the external pudendal artery has been found to increase, coupled with an abrupt and excessive increase in blood input into the mammary gland and increase of mean blood velocity (Barbagianni, 2016).

4. Ultrasonographic examination of the udder in sheep health management Application of ultrasonographic examination of the udder in sheep flocks might be employed as a complementary method in the diagnosis of mammary disorders (Fragkou et al., 2014). However, we consider that, at the moment, despite the difficulties in interpreting the results of somatic cell counts in ewes’ milk (Berthelot et al., 2005), the combination of bacteriological and cytologi-

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Fig. 33. Spectral waveforms of the external pudendal artery (Doppler ultrasonography) obtained 3 weeks before lambing; markedly reduced blood input into the vessel, which has a smaller diametre (for comparison with images obtained later in ® pregnancy or during lactation: Figs. 34, 35). Image taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, Doppler imaging frequency: 6.6 MHz – scanning depth: 60 mm.

Fig. 35. Spectral waveforms of the external pudendal artery (Doppler ultrasonography) obtained the first week after lambing; markedly increased blood input into the vessel (for comparison with images obtained during pregnancy: Figs. 33, 34). Image ® taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, Doppler imaging frequency: 6.6 MHz – scanning depth: 60 mm.

additional information, e.g., regarding presence of abcesses or of increased quantity of fibrous tissue consequently to long-standing mastitis. Such findings can be useful in deciding the management of animals that are found with reduced milk production. Estimation of the dimensions of gland cistern of ewes in the flock would support decisions regarding milking frequency to be applied in a flock. Identification of ewes with gland cistern with increased capacity can be also incorporated in the selection process of animals for eventual improvement of milk production of the flock (Nudda et al., 2000; Castillo et al., 2008; Rovai et al., 2008; Ayadi et al., 2011). Finally, examination of the teat can provide indications regarding optimising use of the milking machine by applying the appropriate settings. 5. Concluding remarks

Fig. 34. Spectral waveforms of the external pudendal artery (Doppler ultrasonography) obtained 2 days before lambing; increase of blood input into the vessel (for comparison with images obtained earlier in pregnancy or during lactation: ® Figs. 33,35). Image taken and processed on a MyLab 30 ultrasonography system (ESAOTE SpA, Genova, Italy) with linear transducer, Doppler imaging frequency: 6.6 MHz – scanning depth: 60 mm.

cal examinations provides an acceptable diagnostic methodology (Fragkou et al., 2014), which, for diagnosis of subclinical mastitis, cannot be replaced by the ultrasonographic examination alone. The technique can have only an ancillary role in the diagnosis of the disease, for example during investigation of cases, in which clinical diagnosis alone can prove of little help, e.g., in animals with small-size, deep mammary nodules. The ultrasonographic examination of the udder of ewes at the end of a lactation period, as part of routine udder examination performed at that point (Fthenakis et al., 2012), would provide

The ultrasonographic examination of the udder of ewes is a useful ancillary technique for in depth study of disorders of the udder. The technique has many uses and can support decisions during a diagnostic and/or health management procedures. However, the technique should be used in combination with established diagnostic methods, including clinical, bacteriological and cytological techniques. This, of course, does not preclude the use of the technique in sheep udder health management; it only underlines that limitations of the technique do occur and these should be taken into account during its application. Conflict of interest The authors have nothing to disclose. References Alejandro, M., Roca, A., Romero, G., Díaz, J.R., 2014a. Effects of overmilking and liner type and characteristics on teat tissue in small ruminants. J. Dairy Res. 81, 215–222. Alejandro, M., Rodriguez, M., Peris, C., Diaz, J.R., 2014b. Study of ultrasound scanning as method to estimate changes in teat thickness due to machine milking in Manchega ewes. Small Rumin. Res. 119, 138–145.

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