Anim. Reprod., v.10, n.3, p.215-222, Jul./Sept. 2013

Doppler ultrasonography as a tool for ovarian management

J.H.M. Viana1,2,5, E.K.N. Arashiro1,2, L.G.B. Siqueira1, A.M. Ghetti3, V.S. Areas4, C.R.B. Guimarães2, M.P. Palhao2, L.S.A. Camargo1,2, C.A.C. Fernandes2

1Embrapa Dairy Cattle Research Center, Juiz de Fora, MG, Brazil. 2University Jose do Rosario Vellano, Alfenas, MG, Brazil. 3Fluminense Federal University, Niterói, RJ, Brazil. 4Espirito Santo Federal University, Alegre, ES, Brazil.

Abstract B-mode ultrasonography, for example, had a central role in the characterization of ovarian follicle dynamics Doppler ultrasonography allows the in different domestic species and in the subsequent characterization and measurement of blood flow, and development of several protocols to control ovarian can be used to indirectly make inference regarding the function for assisted reproductive technologies (ARTs) functionality of different organs, including the ovaries. such as timed artificial insemination, superovulation, Several studies highlighted the importance of an and oocyte pick-up (Adams et al., 2008). The versatility adequate blood flow for follicle development and and the number of potential use have made the B-mode acquisition of ovulatory potential, and for progesterone ultrasound a valuable and widespread adopted tool in secretion by the corpus luteum. Due to some animal reproduction sciences. particularities of the ovarian vascular anatomy, Over the past few decades, various new however, different strategies had to be developed to technologies for image diagnosis with potential use in measure the blood flow detected by Doppler imaging. reproductive medicine emerged, including the Doppler Some of these approaches were successful to ultrasonography (King, 2006). The Doppler technology characterize the patterns of blood flow throughout the allows the identification of movement of cells or tissues estrous cycle, pregnancy, and early postpartum, but and is particularly important in the characterization and require post-acquisition image processing and do not measurement of blood flow. The vascular dynamics is allow real time decisions to be taken. The subjective very closely related to the function of different organs, evaluation of blood flow has been alternatively used in including those from the reproductive tract, and field conditions aiming to early detect non-pregnant consequently is an important evaluation parameter of animals, select embryo recipients, or predict artificial reproductive function. Doppler ultrasonography is insemination and in vitro fertilization outcomes. In routinely used in human gynecology and obstetrics, summary, color-Doppler imaging provides important frequently as the diagnostic gold standard, and also to information about the function of corpus luteum and predict in vitro fertilization outcomes (Chui et al., 1997; follicles, supporting clinical diagnoses and management Borini et al., 2001). The use of Doppler ultrasonography decisions. Nonetheless, the adoption of Doppler in animal reproduction research is more recent but not ultrasonography as a routine exam, instead of a limited less important, and several studies have demonstrated use in an individual basis, requires further development the relationship of blood flow and ovarian and uterine of feasible blood flow evaluation procedures and the function throughout the estrous cycle and pregnancy establishment of reference values. (Acosta et al., 2002, 2003; Miyamoto et al., 2005; Honnens et al., 2008; Herzog et al., 2010). In spite of its Keywords: blood flow, cattle, reproduction, vascular potential usefulness for reproductive management dynamics. (Miyamoto et al., 2006; Matsui and Miyamoto, 2009), and differently from the human medicine, the adoption Introduction of Doppler imaging technology in large animal practice is still limited. The development of image technologies Two main factors have contributed to this low significantly contributed to advances in the field of use of Doppler technology in cattle: 1) the availability biological sciences. The possibility of a non-invasive, and cost of portable devices, and 2) the relative lack of real time approach to assess tissues and organs was of knowledge by practitioners about its potentials as a tool great importance for the study of many physiological to evaluate reproductive function. Along with the recent mechanisms and for the diagnosis of pathological technological advances in electronics and data conditions. For these purposes, several of technologies, processing computers there is a trend for the reduction from the X-ray to the modern computer tomography, in both size and price of portable ultrasound devices have been used in human and veterinary medicine. The (Herzog and Bollwein, 2007). The present article aims ______5Corresponding author: [email protected] Phone: +55(32)3311-7438 Received: May 5, 2013 Accepted: July 15, 2013 Viana et al. Doppler ultrasonography of the ovary. to review some of the particularities of the use of however, requires a well-immobilized patient during the Doppler ultrasonography in the evaluation of ovarian exam, what is frequently difficult to achieve in large function and its implications for cattle reproductive animals. The interference of breathing and peristaltic management, considering the basic mechanisms of the movements may also impair correct caliper positioning technique, the vascular physiology of the ovary, and the during the exam and so spectral mode Doppler is hardly alternatives to evaluate its vascularization. used in routine evaluations of cattle ovaries. Additionally, the Doppler shift is affected by the angle Doppler ultrasonography: basic principles, (insonation angle) between the Doppler beam and the limitations, and alternatives for ovarian evaluation flow direction; therefore calculation of blood flow velocity requires the knowledge of the vessel orientation The Doppler Effect was first described in 1842 (Ginther, 2007). Besides being small, most of the by Christian Johan Doppler. It is a natural phenomenon vessels in the follicular wall or around the corpus characterized by the apparent change in the sound wave luteum are tortuous, and it is difficult to establish the frequency when the source of the wave moves towards insonation angle and, consequently, to have a correct or away from the receptor. The difference between the estimation of flow velocity. Finally, the architecture of frequency generated and received (Doppler shift) is the ovarian artery, which is coiled around the ovarian proportional to the velocity of the movement. This vein, reduces the pulse pattern of the ovarian arterioles, effect also occurs with ultrasound waves, and the affecting PI and RI. frequency difference caused by the movement of blood To overcome these limitations, some cells allows for the detection and measurement of blood alternative approaches have been used for the flow (Ginther, 2007). The change in the frequency of assessment of blood flow in follicles and corpora lutea. reflected waves relative to emitted waves can be One of the most used is the measurement of the colored positive, when blood cells move towards de transducer; area in one or a few representative images of the area or negative, when cells move away from the transducer under study (Acosta et al., 2002; Ginther et al., 2007; (Herzog and Bollwein, 2007). Rauch et al., 2008). In this approach, the ovarian The Doppler shift can be represented in the structure is scanned and the color-Doppler mode is used ultrasound screen as a colored image (color and power to identify the blood flow, and cross section images of Doppler) or as a graph (spectral Doppler). In the color the central area or of the areas with greatest Doppler flow mode, the Doppler shift is shown as a spectrum of signal are recorded and later measured using software one or two colors, usually red and blue, which for image analysis. Alternatively, power mode Doppler represents the direction of blood flow. In the power flow can be used and pixel analysis performed in the colored mode the color represents the intensity of the Doppler areas to have a semiquantitative assessment of luteal or signal, regardless of direction. In the spectral flow follicle blood flow (Lüttgenau et al., 2011a, b). This mode, the Doppler shift is represented as a chart (speed methodology is relatively simple to perform and results by time). In an artery, for example, the spectral graph will are usually coherent with the expected physiological typically have a wave form corresponding to the arterial variation in blood flow (Herzog et al., 2010). pulse in each cardiac cycle (systole and diastole), while in Unfortunately, images post-processing prevents real- veins the flow will be almost constant, without a pulse time decisions to be taken. Although a high repeatability pattern (Herzog and Bollwein, 2007). The spectral flow was reported for blood flow evaluation based on single mode allows a detailed analysis of flow distribution and images (Lüttgenau et al., 2011b), bias may also occur waveform, and consequently the calculation of velocity due to the unevenly distribution of angiogenesis, and indices (pulsatility index [PI], resistance index [RI]), especially in the follicle, where largest vessels are in one particular vessel. The color flow mode, on the present mainly in the basal region (Jiang et al., 2003). other hand, provides less flow information, but allows During transrectal ultrasound exam of the ovaries, it is an overview of the blood flow perfusion in a region. not always possible to adjust the angle of scanning in Most of the objective measurements of blood order to have in the same (“representative”) picture the flow performed by Doppler ultrasonography, including cross section of a follicle on its maximum diameter and calculation of flow velocity and indices, are usually on the area with the maximum number of colored performed in large and straight arteries and veins. pixels. This could explain, to some extent, the relatively However, the evaluation of blood flow in the genital low correlation between follicle blood flow (FBF) and system, especially the ovary may be particularly follicle diameter observed during dominant follicle challenging. Arterioles and venules detectable by growth (Lüttgenau et al., 2011a; Arashiro et al., 2012), ultrasound in follicles and corpora lutea represents only comparing to that observed between corpus luteum part of the vascular plexuses of these structures (Jiang et blood flow (CLBF) and corpus luteum (CL) size. al., 2003), and besides having very small diameters they Another approach routinely used by both show slow blood flow, requiring the Doppler function to researchers and practitioners is the subjective evaluation be set for a high sensitivity (usually by decreasing pulse of Doppler images (Ghetti et al., 2012). In this repetition frequency [PRF]). This high sensitivity, methodology the technician ranks blood flow based on a

216 Anim. Reprod., v.10, n.3, p.215-222, Jul./Sept. 2013 Viana et al. Doppler ultrasonography of the ovary. general overview of the Doppler signal in all areas of flow, using a set of images that covers the whole the structure, based on a mental reconstruction of the structure under analysis. Besides, it is possible to three-dimensional image of the vascularization. When reconstruct the vascular architecture of follicles and there is few Doppler signal, as occurs in small follicles, CL, what was previously done only with invasive blood flow can also be simply classified as “present” or methodologies such as vascular polymer injection and “absent” (Pancarci et al., 2012). The subjective subsequent corrosion of organic tissues (Jiang et al., approach is a fast and straightforward way to evaluate 2003). The 3D modeling approach also requires post- blood flow, in which diagnostic and decisions can be acquisition image processing and therefore is time- taken during the exam and, consequently, is suitable to consuming, but may open new possibilities for ovarian most of the field reproductive management routines. vascular dynamics studies. Figure 2 shows 3D models The subjective approach, as any other methodology of a CL on day 12 of the estrous cycle with a typical based on visual ranking instead of objective distribution of blood flow (from the periphery to the measurement, is more difficult to standardize and center; [2B]), and a CL with an unusual vascular requires previous experience of the evaluator (Siqueira architecture (from the center to the periphery; [2D]). et al., 2013). Moreover, it will be more efficient when Although this uncommon blood flow pattern, the cow the amount of detectable blood flow is larger, as occurs bearing this CL was later diagnosed as pregnant when we evaluate blood flow in the CL, in comparison (personal data). to evaluation in follicles; or when the expected differences are greater, as occurs in day-20 CL between pregnant and nonpregnant cows. Previous studies, however, reported a good correlation of blood flow between objective and subjective methods, for both follicles and CL (R = 0.72 and R = 0.78, respectively; P < 0.0001), in a single evaluation (Areas et al., 2012; Ghetti et al., 2012). Figure 1 illustrates the subjective and objective approaches to evaluate the CL.

Figure 2. Three-dimensional modeling of corpus luteum vascularization. A-D: Color flow Doppler imaging (A, C) and respective 3D models (B, D) of a corpus luteum with a typical distribution of blood flow (from the periphery to the center; A, B), and of a corpus luteum with an unusual vascular architecture (from the center to Figure 1. Corpus luteum blood flow evaluation with the periphery; C, D). color flow Doppler imaging. A-C: Corpus luteum subjectively scored as presenting low (A), intermediate Computer-assisted ultrasound image analysis (B) or high (C) vascularization. D: Corpus luteum with was first reported for the evaluation of CL and follicle Doppler signal (colored) area delimited (in white) for echotexture (Singh et al., 2003), and is a requirement further objective measurement with image assisted for most studies on the objective blood flow evaluation computer analysis. in both 2D and 3D Doppler imaging. Different computer programs were used in ovarian Doppler Three-dimensional (3D) modeling was evaluation, including open-source (Image-J, Acosta et alternatively used to objective evaluation of blood flow al., 2002) and commercial versions of general purpose volume in corpora lutea (Jokubkiene et al., 2006) and (Adobe Photoshop, Acosta et al., 2002; FixFoto, follicles (Lozano et al., 2007) in human and, more Lüttgenau et al., 2011b; Mimics, Arashiro et al., 2012) recently, in cattle (Arashiro et al., 2012). This new or custom image analysis software (PixelFlux, Rauch et approach allowed an objective measurement of blood al., 2008; VOCAL, Jokubkiene et al., 2006). Software

Anim. Reprod., v.10, n.3, p.215-222, Jul./Sept. 2013 217 Viana et al. Doppler ultrasonography of the ovary. choice must consider the goals and complexity of each of the CL, particularly during luteolysis, when changes study, the Doppler ultrasound device available, as well in luteal tissue volume are slower and more progressive as the type and resolution of the image files generated. than in luteal function (Niswender et al., 1994). Coherently, the correlations between CLBF or Vascularization and ovarian function progesterone and CL size are not as strong as between CLBF and plasma progesterone, demonstrating that The development of the ovarian follicle is Doppler ultrasonography improves the accuracy of closely related to the development of a vascular network diagnoses by adding physiological information from the in the theca layer, which provides nutrients, hormones organ (Herzog et al., 2010). Interestingly, both natural and oxygen to support follicle growth and and PGF2α-induced luteolysis are characterized by an steroidogenesis. There are no blood vessels in the initial acute increase in blood flow, followed by a granulosa layer and metabolites reach the intrafollicular significant drop 24 to 48 h later (Ginther et al., 2007). environment by diffusion. The vascular plexus of This initial rise is associated with a transient medium and large follicles are supplied by individual vasodilation in peripheral vessels of the CL, driven by spiral arteries but drained by several veins (Jiang et al., an increase in endothelial nitric oxide synthase 2003). There are many evidences demonstrating the stimulated by the PGF2α (Shirasuna et al., 2008), and relationship between vascularization and follicular must be taken into account as a factor affecting single function. Angiogenesis and ischaemia are related to the evaluations of the CLBF performed during the expected fate of follicles, although it is not clear whether they are period of luteolysis, specially when the goal is to early cause or consequence of follicle development or atresia identify non-pregnant animals (Siqueira et al., 2013). (Jiang et al., 2003). Blood flow affects the composition of follicular fluid (Rodgers and Irving-Rodgers, 2010) Color flow Doppler imaging in the reproductive and is positively correlated to intrafollicular estradiol management concentrations and estradiol:progesterone ratios (Pancarci et al., 2012). Follicular blood flow increases Color flow Doppler imaging can be used to during follicle deviation (Arashiro et al., 2012) and assess the amount and pattern of blood flow within the reaches a peak in the preovulatory follicle after the ovary, and indirectly indicates its functionality. This is onset of the LH surge or 0.5 h after GnRH injection particularly useful to characterize the presence of luteal (Acosta et al., 2003), supporting the concept that a close tissue, as in early developing CL, tumors, or in association between blood flow and the morphological luteinized cysts (Rauch et al., 2008), and to make and functional changes precedes ovulation. indirect inference regarding CL function, i.e., Consequently, FBF affects oocyte quality and its progesterone secretion. Visualization of CLBF may developmental potential (Sutton et al., 2003). Follicle therefore be used either in studies of ovarian physiology vascularization is also associated to responsiveness to or for reproductive management decisions. A CL that exogenous stimulation and to further embryo does not present enough Doppler signals (colored implantation rates (Lozano et al., 2007), and has been pixels), an evidence of lack of blood supply, may be used in human medicine to predict in vitro fertilization considered as not functional; the same CL, however, outcomes (Chui et al., 1997). may present a normal size (diameter or area). From this The CL is highly vascularized and shows the point of view, gray-scale ultrasonography is limited to greatest blood flow per unit of tissue in the body differentiate from a functional vs. a nonfunctional CL, if (Acosta et al., 2002). Luteogenesis is characterized by they have similar size. This type of assessment of intensive angiogenesis, and CLBF increases 3 to 4-fold functionality is only possible by using Doppler imaging. in the first 96 h after ovulation (Acosta et al., 2003). Indeed, blood flow has been suggested to be more Most of steroidogenic cells are adjacent to one or more appropriate than size for CL function evaluation capillaries (Reynolds et al., 2000) and luteal blood flow (Herzog et al., 2010), because CL vascularization plays perfusion is so intense that affects acoustic impedance a key role in regulating luteal function (Miyamoto et al., of the organ, resulting in the hypoechoic echotexture 2005). Evaluations of CLBF could, consequently, be characteristic of the CL (Singh et al., 1997). useful to detect pregnancy failures or to predict Neovascularization during CL development is important pregnancy rates after embryo transfer or timed-AI. to support the rapid growth of luteal tissue and is In cattle and other domestic mammals, a essential for both the provision of substrate for functional CL is required for pregnancy establishment, progesterone biosynthesis and for the delivery of because progesterone supports preimplantation steroids to the general circulation (Miyazaki et al., embryonic development, allowing maternal recognition 1998). Plasma progesterone concentrations are of pregnancy (Mann and Lamming, 1999). Failure in positively correlated to CLBF throughout the estrous conception or the presence of a non-viable embryo cycle, reflecting the development and regression of results in absence of maternal recognition of pregnancy, luteal tissue (Herzog and Bollwein, 2007). Progesterone which will consequently lead to CL regression secretion is the gold standard for functional evaluation (reviewed by Niswender et al., 2000). Luteolysis is

218 Anim. Reprod., v.10, n.3, p.215-222, Jul./Sept. 2013 Viana et al. Doppler ultrasonography of the ovary. primarily associated to a progressive decrease in the early resynchronization of nonpregnant animals and blood flow to the CL in response to prostaglandin F2α consequent reduction in days open and calving intervals. and luteal oxytocin. Although the CL´s loss of function In embryo transfer programs, synchronized (progesterone secretion) responds rapidly to the recipients are routinely selected based on the presence decrease in blood flow, its morphological regression and quality of the CL on days 7 or 8 of the estrous (reduction in size) only becomes evident hours later cycle. The relationship between CL quality, assessed by (Niswender et al., 1994). This asynchrony between size or echotexture, and pregnancy rates have been functional and morphological changes limits the evaluated after the transfer of embryos produced in vivo usefulness of CL evaluation by conventional B-mode or in vitro (Siqueira et al., 2009). Although there is a ultrasonography, based on the evaluation of CL size general consensus that CL function affects the (diameter or area), to determine luteal function around likelihood of pregnancy, the relatively poor association the time of luteolysis (Kastelic et al., 1990). The use of between CL morphological characteristics and color-Doppler ultrasound can overcome these progesterone production limits the predictive value of limitations because it allows real-time CLBF B-mode ultrasonography for recipient selection. assessment, an indirect measure of CL functionality, Progesterone secretion and CL blood flow are positively especially by the end of the estrous cycle (Miyamoto et correlated throughout the estrous cycle (Herzog et al., al., 2006; Herzog et al., 2010). In the reproductive 2010) and therefore color-Doppler ultrasonography management routine, an important strategy to improve could be a valuable tool to select embryo recipients. Our reproductive efficiency is to detect failure in conception preliminary results (Guimarães et al.; Universidade José as early as possible. It has been suggested that color- do Rosário Vellano, Alfenas, MG; unpublished data) Doppler imaging could be used to early detect however, showed no differences in CLBF between nonpregnant animals, by evaluating CLBF (Matsui and embryo recipients later diagnosed as pregnant or Miyamoto, 2009; Siqueira et al., 2013). nonpregnant. This apparent contradiction may be caused Although gray-scale ultrasonography has been by the complex interplay among embryo quality, successfully used for transrectal early pregnancy embryo developmental stage, and endometrial timing by diagnosis in cattle (Pierson and Ginther, 1984), this progesterone (Lonergan, 2011). Although the role of approach presents low reliability and accuracy when progesterone in the establishment and maintenance of performed earlier than 26 days post AI (Pieterse et al., pregnancy is well known (Mann and Lamming, 1999), 1990) because clear visualization of the embryo/vesicle the minimum threshold for the establishment of in the uterine lumen at this stage is difficult and time pregnancy is controversial (Spell et al., 2001) and consuming (Quintela et al., 2012). Therefore, consequently it is difficult to determine a cutoff value. considering that a functional CL is mandatory for Moreover, in spite of characterizing the association of pregnancy establishment, the lack of CLBF on specific CLBF and progesterone during the estrous cycle days of the estrous cycle may be used as a direct (Herzog et al., 2010), the same group observed that indicator of nonpregnancy. In fact, previous studies single Doppler evaluations during mid-luteal phase (on have already suggested that a decrease in CLBF around day 9 of the estrous cycle) failed to correlate CLBF and 19 to 21 days post AI is an indicator of nonpregnancy plasma P4 concentration (Lüttgenau et al., 2011b). (Matsui and Miyamoto, 2009) and also that color Thus, it is likely that Doppler evaluation, as occurs with Doppler flow imaging could be useful for an accurate CL size and even with progesterone concentration, will early pregnancy diagnosis in cattle (Matsui and provide additional information to interpret further Miyamoto, 2009; Quintela et al., 2012). Briefly, the pregnancy rates after embryo transfer, but may be of idea is to detect, by color-Doppler imaging, normal- limited use as a tool to select recipients. sized, non-functional corpora lutea, which indicates Follicular blood flow evaluation has been used failure in conception. Results of these types of to predict follicle status and oocyte quality in human approaches are still controversial, and some authors medicine (Sutton et al., 2003; Lozano et al., 2007). In recommend CLBF for early pregnancy diagnosis cattle, there are some studies highlighting the (Matsui and Miyamoto, 2009; Quintela et al., 2012) but importance of vascularization on follicle viability, others reported low accuracy (Utt et al., 2009) and growth, ability to become dominant and ovulate (Acosta reliability (Herzog et al., 2011) of this diagnostic et al., 2003; 2005; Pancarci et al., 2012), but few reports method. Perhaps, the explanation for this apparent associating vascularity of preovulatory follicles and disagreement lays on the fact that CLBF evaluation has subsequent fertility (Siddiqui et al., 2009a). Although been performed at different days of the estrous cycle in evidences of a direct relationship between blood flow those studies. A recent report showed high accuracy and and the fate of the follicle were previously reported reliability of pregnancy diagnoses performed on day 20 (Acosta et al., 2005), the use of FBF in a reproductive post AI, using subjective evaluation of CLBF as an routine still requires the establishment of feasible unique criterion (Siqueira et al., 2013). The subjective evaluation procedures and respective reference values or evaluation of CLBF is a feasible approach, and its criteria. For example, FBF can be detected from small adoption in the reproductive routine may allow for the (4-6 mm) to preovulatory follicles and this information

Anim. Reprod., v.10, n.3, p.215-222, Jul./Sept. 2013 219 Viana et al. Doppler ultrasonography of the ovary. can be used to predict oocyte quality and in vitro Arashiro EKN, Palhao MP, Santos JRL, Fontes R, embryo production (Siddiqui et al., 2009b; Pancarci et Henry M, Viana JHM. 2012. Three dimentional al., 2012). In the commercial embryo production modeling of color Doppler ultrasound images: a new laboratories, however, oocytes are not tracked approach to the evaluation of follicular vascularization individually throughout the process and therefore in cattle. Reprod Domest Anim, 47:489. (abstract). embryos cannot be linked to the status of the follicle of Areas VS, Siqueira LGB, Ghetti AM, Souza ED, origin. A single exam of FBF can be performed in Arashiro EKN, Camargo LSA, Viana JHM. 2012. preovulatory follicles, for example as selection criteria Evaluation of corpora lutea vascularization by color to decide for insemination. Follicles presenting no Doppler during the period of maternal recognition of Doppler signal are probably undergoing atresia (Acosta pregnancy: preliminary data. Anim Reprod, 9:673. et al., 2005) and will be related to conception failures. (abstract). In most cases, however, preovulatory follicles will Borini A, Maccolini A, Tallarini A, Bonu MA, present some degree of vascularization (Ghetti et al., Sciajno R, Flamigni C. 2001. Perifollicular vascularity 2012) and the possibility of using these differences to and its relationship with oocyte maturity and IVF predict fertility is controversial. Actually, there is a outcome. Ann NY Acad Sci, 943:64-67. significant variation in other predictors of follicle status, Chui DKC, Pugh ND, Walker SM, Gregory L, Shaw such as in intrafollicular concentrations of estradiol, RW. 1997. Follicular vascularity - the predictive value even among growing follicles of the same diameter and of transvaginal power Doppler ultrasonography in an in presenting estradiol:progesterone ratios higher than 1 vitro fertilization programme: a preliminary study. Hum (Viana et al., 2013). The physiological relevance of this Reprod, 12:191-196. variation is unclear, and consequently the same is Ghetti AM, Siqueira LGB, Areas VS, Arashiro EKN, possible for minor differences in FBF. Fernandes CAC, Palhao MP, Viana JHM. 2012. In summary, color Doppler imaging provides Comparison of objective and subjective methods for important information about the function of CL and evaluating the vascular pattern of the preovulatory follicle follicles, supporting clinical diagnoses and management using color Doppler. Anim Reprod, 9:657. (abstract). decisions. For example, the technology can be used for Ginther OJ (Ed.). 2007. 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