Theriogenology 81 (2014) Contents lists available at ScienceDirect. Theriogenology. journal homepage:

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1 Theriogenology 81 (2014) Contents lists available at ScienceDirect Theriogenology journal homepage: 40 th Anniversary Special Issue How ultrasound technologies have expanded and revolutionized research in reproduction in large animals O.J. Ginther a,b, * a Eutheria Foundation, Cross Plains, Wisconsin, USA b Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA article info Article history: Received 25 June 2013 Received in revised form 6 September 2013 Accepted 7 September 2013 Keywords: Arterial pulse Color-Doppler imaging Doppler ultrasonography Gray-scale imaging Vascular perfusion Gray-scale ultrasonic imaging (UI) was introduced in 1980 and initially was used to examine clinically the reproductive tract of mares. By 1983 in mares and 1984 in heifers/ cows, UI had become a tool for basic research. In each species, transrectal gray-scale UI has been used extensively to characterize follicle dynamics and investigate the gonadotropic control and hormonal role of the follicles. However, the use of transrectal UI has also disclosed and characterized many other aspects of reproduction in each species, including (1) endometrial echotexture as a biological indicator of circulating estradiol concentrations, (2) relative location of the genital tubercle for fetal gender diagnosis by Days 50 to 60, and (3) timing of follicle evacuation during ovulation. Discoveries in mares include (1) embryo mobility wherein the spherical conceptus (6 16 mm) travels to all parts of the uterus on Days 11 to 15, (2) how one embryo of a twin set eliminates the other without self-inflicted damage, and (3) serration of the granulosum of the preovulatory follicle opposite to the future rupture site as an indicator of imminent ovulation. Studies with color-doppler UI have shown that vascular perfusion of the endometrium follows the equine embryo back and forth between uterine horns and follows the expansion of the bovine allantochorion throughout each horn. In heifers, blood flow in the CL increases during the ascending portion of an individual pulse of PGF2a metabolite and then decreases. These examples highlight the power of UI in reproduction research. Without UI, it is likely that these and many other findings would still be unknown. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction I wish I could see what s going on in there, was a lament of farm animal reproductive biologists and clinicians throughout most of the 20th century. During the 1980s, gray-scale (B-mode) ultrasonic imaging (UI) of the reproductive organs in horses and cattle revealed dynamic yet unexpected events (e.g., intrauterine travels of the equine embryo, acrobatic feats of the equine and bovine fetus). Before the availability of UI, such activities were not on the researcher s menu or could not be studied without worrying about invasive interference. The physical nature of known events could be contemplated but not described (e.g., * Corresponding author. Tel.: þ ; fax: þ address: ginther@vetmed.wisc.edu. timeline and characteristics of follicle evacuation during ovulation). This review discusses the early history and eventual lofty status of ultrasound technologies in reproduction research. Mares and, a few years later, heifers/cows provided the incentive for the initial adaptation and enthusiasm for grayscale UI. The premier event (as in Columbus discovering the New World) was a report in Theriogenology in 1980 by Palmer and Driancourt [1] on UI of the reproductive organs of the mare. The report was published a century after the discovery of the piezoelectric effect of certain crystals [2]. Crystals in ultrasound transducers have the piezoelectric characteristic of expanding and contracting in response to alternating polarities of electric signals. The crystals in the transducer produce ultrasound waves when applied to tissue and receive and convert the resulting echoes back into X/$ see front matter Ó 2014 Elsevier Inc. All rights reserved.

2 O.J. Ginther / Theriogenology 81 (2014) electric signals. The signals are converted and displayed as shades of gray that represent the intensity of the echoes and the locations of the tissue reflectors. Sounds simple. Many research findings in large animal reproduction during the past 30 years can be credited entirely to grayscale UI. The literature has become so voluminous on horses and cattle that some of the sections in this review have been reduced to a selected list of ultrasound-based research contributions that illustrate the research power of ultrasound technologies in reproductive biology; the lists are not comprehensive. In addition, preference is given to the apparent original reports for historical purposes. The review heralds examples of the capability of UI for data collection and statistical testing of hypotheses by researchers and as a source of enlightenment and fascination for all who watch the moving images. It is also entertaining to see something for the first time, even when it is an illusion (Fig. 1). Equine embryo mobility is featured as an example of a discovery that was solely a product of transrectal gray-scale UI. The discovery can be considered fundamental in that it extended into unexpected areas. Consideration will also be given to research findings by gray-scale UI in females of large species other than horses and cattle, specialized research techniques, handling of research data, and the application of UI to research in male genitalia. Color-Doppler UI and Doppler ultrasonography are also discussed. The term ultrasonography refers exclusively to the production of Doppler graphs that represent the changing blood velocities within an arterial pulse and therefore will be distinct from color Doppler UI. Applied and diagnostic uses of UI in reproductive management in horses, cattle, and other large domestic species [3 12] and the principles, equipment, and techniques [2,4,13] have been well-reported and are not considered. The review updates and expands on previous reviews on research applications of gray-scale UI in horses and cattle [2,3,6,14,15]. This review is part of the 40-year commemorative issue of Theriogenology and notes the role of the journal in disseminating the initial (Table 1) and mounting reports on UI and ultrasonography. 2. Gray-scale UI in mares In the early 1980s, there was a rapid increase in transrectal gray-scale UI by equine theriogenologists [16 19]. Horse-breeding farms were a natural site for initiation of the technology for several reasons: (1) intensive veterinary management of reproduction in individual mares, (2) justification for purchase of relatively expensive equipment, and (3) fluid-filled embryonal and fetal conceptus and the noncoiled shape of the uterus permitted intriguing displays of the dynamic interactions between the conceptus and uterus. The transrectal route for insertion of the ultrasound transducer in the early years came naturally because of the well-established transrectal route for routine tactile examination of the internal reproductive organs in mares. The linear intrarectal transducers that were used for mares in 1980 to 1982 were of low frequency (e.g., 3.0 MHz). The low resolution of the original transducers is apparent in published Polaroid ultrasonograms of equine embryos from Days 11 to 48 [20]. In 1983, a 5.0-MHz linear transducer was introduced [13]. The 5.0-MHz transducer had less depth of penetration, but the greater resolution yielded detailed information on structures close to the Table 1 Apparent milestones in the introduction of ultrasound technologies for research study of the reproductive organs in large animals. a Fig. 1. Examples of image illusions that serve to enliven discussion among observers. (A) Cross sections of the rear hooves of a 200-day equine fetus and the surrounding tissues of the uterine horn. (B) Segment of the umbilical cord of a 70-day equine fetus. The echoic area below the poodle is a portion of the fetus. (C) The agonizing ghostly face is an equine CL. (D) The arrows delineate a cross section of the pelvic urethra of a stallion during ejaculation. Dilations of the vesicular-gland ducts (eyes) and urethral lumen (mouth) occur rhythmically with each ejaculatory pulse. Adapted from [2,3] with permission. Year Ultrasound technology b 1980 UI of ovaries and uterus in mares [1] MHz linear-array transrectal transducer [13] 1984 UI of ovaries, uterus, and conceptus in heifers [71 73] 1987 UI of genitalia in stallions [187] 1988 UI of genitalia in bulls [191,193] 1989 UI of ovaries and uterus in llamas [117] 1991 UI of ovaries and uterus in zoo animals [126] UI of follicle dynamics in ewes [111], goats [114], sows [115], and water buffalo [122,123] 1998 Ultrasonography of uterine artery in mares [164] 2000 Ultrasonography of uterine artery in cows [165] 2002 Color-Doppler UI of ovaries in cows [169] 2004 Color-Doppler UI of ovaries in mares [171,174] Gray-scale UI, except for 2002 and Abbreviations: UI, ultrasonic imaging. a Some of these research milestones were preceded by reports on the clinical use of ultrasound for pregnancy diagnoses in sheep, goats, and pigs (reviewed in [12]). b References in italics indicate publication in Theriogenology.

3 114 O.J. Ginther / Theriogenology 81 (2014) transducer and separated only by the wall of the rectum. Consequently, published ultrasonograms of the echotexture of reproductive structures of mares were superior to those that were available for women, who at the time were being scanned through the abdominal wall [2]. Subsequently, this disadvantage was negated with the adaptation of transvaginal transducers for women. The ultrasound scanners used in the early 1980s had analog scan converters; therefore, filming the scans was not a simple option [2]. By 1986, digital scan converters became available and recording the scans and assessing the scans by computerized image analyses increased the research power of UI. Equine reproductive physiologists were alerted to the tremendous potential of gray-scale UI for their own studies by the original publication in 1980 [1] but were frustrated by equipment cost. Our laboratory was fortunate to be given the loan of a high-quality ultrasound scanner with an analog converter in The scanner screen became a window to knowledge for our laboratory. Approximately 35 publications (63% in Theriogenology) from 1983 to 1985 based entirely on gray-scale UI contributed the following examples of original research findings: (1) UI dynamics of the follicles and CL throughout the estrous cycle [21]; (2) UI anatomy and pathology of the uterus [22]; (3) echotexture of the endometrial folds as an indicator of estrogen exposure for detecting the preovulatory period (Fig. 2) [22,23]; (4) traveling of the spherical (6 16 mm) embryonic vesicle to all parts of the uterus (embryo mobility) on Days 11 to 14 or 15 (Day 0 ¼ ovulation) [24 26] and fixation on Day 15 or 16 [19] in ponies and horses, respectively; (5) interactions between twins during embryo mobility [27]; (6) a technique for manual elimination of one member of a twin set during embryo mobility [28]; (7) physical nature of embryonic death [29]; and (8) postfixation mechanism for spontaneous embryo reduction of one member of a twin set (Fig. 3) [30]. The knowledge windfall culminated in the first text and reference book on gray-scale UI in animals in 1986 under the narrow title of Ultrasonic Imaging and Reproductive Events in the Mare [31]. It was the most exciting 3 years of my ongoing 50 years of research. Ultrasound-based contributions in mares after the 1986 book included many studies on ovarian follicles. After the initial characterization of follicle population dynamics based on diameter groupings [21], the technique of identifying individual follicles from examination to examination was introduced by researchers in New York [32]. Monitoring follicles became essential for research on the hormonal mechanisms associated with follicular waves, selection of a dominant follicle, and hormonal preparation of the ovulatory follicle [33 35]. Follicle dynamics were characterized for the prepubertal stage [36], ovulatory season [35,37], anovulatory season [38], during old age [39], and in comparison of miniature ponies and larger breeds [40]. It was also found that the characteristics of the ovulatory follicular wave are surprisingly similar between mares and women, except for the 2.2-fold larger follicle in mares at specific events during a wave [41,42]. A major advance was the finding that both minor waves (waves without a dominant follicle) and major waves occur in mares [43], and the emergence of each wave type is attributable to a surge in FSH concentration [44]. Original ultrasound-based findings in mares that did not involve follicular waves after the 1986 book included: (1) first noninvasive characterization and quantification of uterine contractions in any species [45]; (2) factors affecting contractility and role of uterine contractions in embryo mobility [26]; (3) first noninvasive continuous characterization and timing of Fig. 2. Contrast between estrus and diestrus in the echotexture of uterine horns in mares (cross sections) and heifers (longitudinal sections). The images in heifers are from the curvature at the cranial portion of a horn; the arrows delineate the mesometrial attachment. Adapted from [3,6] with permission. Fig. 3. Process of embryo reduction in twin equine embryos with fixation in the same uterine horn. On Day 17, the thick vascular wall (yellow line) of the yolk sac of the doomed vesicle (Y2) is forced by uterine massage into the thin nonvascular wall of the yolk sac of the thriving vesicle (Y1); the vascular area (red line) of Y1 is shown in contact with the endometrium. Similarly, on Day 29 the vascular wall of the doomed vesicle (yellow line) is blocked from the endometrium. Allantoic sac (A1), yolk sac (Y1, Y2), and embryo proper (E1, E2) are indicated. 1 ¼ thriving vesicle, 2 ¼ doomed vesicle. Adapted from [65] with permission.

4 O.J. Ginther / Theriogenology 81 (2014) follicle evacuation during ovulation in any species [46]; (4) development of fetal gender diagnosis on Days 50 to 60 by relative location of the genital tubercle (Fig. 4) [47]; (5) development of the deprivation concept, whereby an embryo (before Day 40) can eliminate its twin and not suffer selfinflicted damage [48]; (6)changes in echotexture,especially that an anechoic layer of the follicle wall distinguishes the future dominant follicle before the diameter manifestation of follicle selection (deviation) [49]; (7) discovery of serration of the granulosa layer of the preovulatory follicle and its use for predicting imminent ovulation [50]; (8) incidence and nature of embryonic death, including expulsion through the cervix [29,51]; (9) characterization of the natural outcome of fetal twins and in vivo identification of fetal twins in late gestation by transrectal UI of the apposition of twin fetal membranes [52]; (10) effects of ultrasonically detected uterine pathology (e.g., uterine cysts, small intraluminal fluid collections) on fertility [53] and the conclusion that a small fluid collection (diameter, 5 20 mm) during diestrus and detectable only by UI is an indicator of metritis [29,53];(11)incidence and nature of dysorientation of the embryo proper and spontaneous correction [54]; (12) accumulation of fluid in the infundibulum during the estrous cycle [55]; (13) effects of age on uterine function [56]; (14) assessment of fetal fluids, placental thickness [57], and ascending placentitis [58]; and(15) discovery that fetal (after Day 40) activity (Fig. 5) involved frequent changes in orientation and in location among the uterine horns and body, allantoic fluid shifts throughout the uterus, closing and opening of parts or all of each uterine horn, and closure of both horns at mid-pregnancy with both fetal hind limbs encased by the umbilical cord horn [59 61]. Excluding the many research reports involving follicle dynamics, 24 findings or characterizations in mare reproductive physiology are listed that likely would still be unknown or unclarified if it were not for the noninvasive but powerful ultrasound technology of transrectal gray-scale UI. These findings and techniques give credence to the opinion enunciated in the title of this review. In addition to monitoring follicles and detecting ovulation, nonfollicular research Fig. 4. Frontal views across the front limbs (FL), hind limbs (HL), umbilical cord (UC), and tail (T) of a bovine fetus at approximately Day 60. The position of the genital tubercle (GT) is between the hind limbs in the male fetus and caudal to the hind limbs in the female fetus. Adapted from [6] with permission. Fig. 5. Examples of fetal mobility in a mare and heifer. The equine fetus is shown with head (HD) and chest (CH) on the allantoic floor and 4 s later being propelled into the allantoic fluid by a vigorous neck (NK) arch. The amniotic membrane is indicated by arrows. The bovine fetus has done a flipflop from side to side as indicated by the rumen (RU) changing from the upper to lower position. Adapted from [3,6] with permission. findings by UI account for the availability of techniques that canbeusedbyequineclinicians.theseareindicatedaboveby points 3 and 6 for 1983 through 1985 and points 4, 7, 9, and 10 during and after Equine embryo mobility Some of the discoveries in mares that are attributable to transrectal UI can be considered fundamental. For example, the demonstration of equine embryo mobility (Fig. 6), wherein the embryo travels to all parts of the uterus an equivalent of 10 to 20 times per day on Days 11 to 15 [25,26] was a stark departure from the assumption that the embryo at this time was nestled snugly among the endometrial folds. As the encapsulated embryo moves, it carries messages for interaction with the uterine wall (Fig. 6). One message stimulates myometrial contractions, which provide the propulsive force for the embryo [26,62], and another stimulates an increase in vascular perfusion in the uterine wall that moves approximately every 0.5 hour from horn to horn with the embryo [63]. The essentiality of embryo mobility involves a message that is carried to all parts of the uterus to block the uterine luteolytic mechanism, ensuring the required maintenance of the progesterone-producing CL [64]. All of the uterus must be visited by the relatively small embryo because in this species the luteolytic signal from uterus to CL is through systemic channels [65]. On the days of mobility, a substance is distributed that gradually stimulates increased uterine tone [62,66]. By the time the blockage of luteolysis is complete

5 116 O.J. Ginther / Theriogenology 81 (2014) Fig. 6. Sequence (1 4) of events associated with equine embryo mobility, uterine contractions, blockage of luteolysis during the first luteal response to pregnancy, decrease in uterine diameter from an increase in tone, and fixation at a flexure in a caudal uterine horn. Adapted from [162] with permission. (mean, Day 16), the embryonic vesicle has grown and uterine tone has increased (decreased uterine diameter) to the point that fixation occurs at the flexure in a caudal horn that offers the greatest impediment to continued mobility [26,67]. Embryo mobility also provided explanation or rationale for hypotheses on several phenomena that were perplexing to equine veterinarians and biologists [68], including: (1) lack of agreement between side of ovulation and side of fixation (embryo mobility involves both horns with no affect of side of CL), (2) preferential fixation in postpartum mares in the formerly nongravid horn (nongravid horn is smaller), (3) fixation almost always in the caudal portion of one of the uterine horns (bend or impediment to mobility in caudal horn), (4) fixation 1 day earlier in ponies than in horses (uterus is smaller in ponies but embryonic vesicle is not), and (5) greater incidence of unilateral than bilateral fixation of twins (first embryo to become fixed is an impediment to mobility by the other). Ultrasound scanners were the window to the phenomenon of embryo mobility and the means for characterizing the underlying mechanisms. Furthermore, embryo mobility and fetal activity and interactions with the uterus are spectacular events that inspire awe and respect for animal biology. With regard to the impact on laymen as well as professionals, a survey by EQUUS magazine credited embryo mobility as an outstanding discovery in equine research [69]. 4. Gray-scale UI in heifers/cows The first reports on transrectal gray-scale UI for examining the reproductive tract of heifers/cows appeared in a report from France on pregnancy diagnosis in 1982 [70] and reports on the echotexture of the reproductive organs and conceptus appeared in Theriogenology in 1984 [71 73]. Apparently, the greatest current use of UI in cattle in most laboratories is to determine the time of ovulation as a reference point or end point and to diagnose pregnancy. However, the technology has many more uses and potential. Detailed studies have been done on follicle dynamics before puberty [74], during the estrous cycle [75], during early [76] and late pregnancy [77], and during superstimulation [73]. The technology has become essential for collection of follicle data in cattle, as for mares, especially by maintaining the identity of follicles from examination to examination [78]. The follicles have been studied during a wide array of research projects involving many aspects of follicle dynamics, waves, and selection [33,79]. Studies with follicles have included the demonstrations that the emergence of each follicular wave during the estrous cycle [80] and throughout pregnancy [77] is associated with a surge in FSH and that a close two-way coupling is involved in the follicle/fsh relationship [81,82]. A recent study used transrectal UI of follicles to study roles and controls of the preovulatory and periovulatory FSH surges [83]. Echotexture reflects the function and endocrine status for the follicular antrum and wall and for the CL [84 86]. Characterization of the nature and timing for evacuation of approximately 90% of the antral fluid during ovulation involved 4 s [87], compared with 60 s in mares [46]. Studies on follicle dynamics and selection have included other bovine species such as Bos indicus [88] as well as B taurus; for example, deviation in the first postovulatory wave in Nelore heifers and cows occurs approximately 1 day earlier when the future dominant follicle is 2 mm smaller than in Holsteins. Research findings in cattle based on transrectal gray-scale UI have included nonfollicular structures and events as follows: (1) changes in size and echotexture of the CL [73], leading to comparisons of functional and structural CL changes [89] and to the use of UI to define a change in CL area (cm 2 ) as a reference point for initiating experiments [90];(2) ultrasonic appearance of the uterus, including a more heterogeneous echotexture during estrus (Fig. 2) [91]; (3) reduced curling of the uterine horns during estrus [91], unlike the apparent curling during estrus when based on transrectal palpation; (4) characterization of uterine contractions during the estrous cycle and pregnancy [92]; (5) pregnancy characterization [72,93], including detailed descriptions of the conceptus on Days 10 to 60 [94 96]; (6) nature of embryonal and fetal loss [97 99] and factors involved in the loss [100]; (7) determining fetal sex by relative location of the genital tubercle (Fig. 4) [101] and later by identification of genitalia [102]; (8) transient filling and emptying of various segments of the uterus by the fluid-filled placental membranes, gradual increase in intrinsic activity of the fetus between Days 40 to 60, and changes in recumbency, presentation (cranial vs. caudal direction), and intrauterine locations throughout pregnancy (Fig. 5) [6]; (9) assessing uterine inflammation [103] and postpartum ovarian activity and uterine involution [104,105]; (10) detection of twins [99] and spontaneous loss [106], and ultrasound-guided reduction of one member of a twin set [107,108]; and (11)

6 O.J. Ginther / Theriogenology 81 (2014) discovery that several fetal anomalies can be detected by UI on Days 55 to 120, including compact fetal mass, schistosomus reflexus, and fetal hydrops [6]. Early detection (e.g., during fetal gender determination) and termination of the pregnancy is an advantage of UI in that many of these anomalies result in dystocia at term. Although bovine researchers and clinicians were slower than their equine counterpartstoembracegray-scaleui,theeventualresults further entrench the accolades in the title of this review. 5. Gray-scale UI in other large species The experiences with horses and cattle encouraged the use of transrectal UI for evaluating the reproductive organs in sheep, goats, pigs, llamas, water buffalo, and nondomestic large animals. The transabdominal route may be used in smaller species (e.g., sheep), especially for pregnancy diagnosis [2], but the resolution of transrectal UI is needed for detailed studies. For transrectal UI, a transducer extension or a person with a small arm may be needed for rectal insertion and manipulation of the transducer [2]. An extension may also be used for very large animals (e.g., elephants). In sheep, UI has been used for diagnosing and studying pregnancy [109,110], assessing ovarian structures [108,109], studying the association between emergence of follicular waves and FSH surges [111], and determining follicle dynamics with high and low ovulation rates [112]. Pregnancy and follicular waves also have been characterized by UI in goats [113,114] and pigs [115,116]. Earlier studies in llamas demonstrated: (1) echotexture and morphology of the reproductive organs, including straightening and curling of the uterine horns during estrus and diestrus, respectively [117]; (2) follicular waves, including growth and regression of individual follicles [118]; and (3) echotexture of the CL [119]. Subsequent studies in Canada in llamas have utilized UI as an aid in transvaginal collection of oocytes [120] and for a series of studies that culminated in the first demonstration of an ovulation-inducing factor in the seminal plasma of llamas, alpacas, and bulls [121]. Follicular waves have also been shown by UI to occur in water buffalo [122,123] and have implications for assisted reproduction [124]. Although slow to be adopted, transrectal scanners are without parallel in the study of the reproductive organs of nondomesticated large animals. The knowledge gained on the reproductive system through transrectal UI and other ultrasound techniques (e.g., oocyte collection) have shown potential in aiding these species to propagate, especially those that are endangered. In farmed red deer, transrectal UI was used in 1990 to establish criteria for predicting calving data by measurements of the uterus, amniotic sac, placentomes, and several parts of the fetus [125]. The introductory publication on transrectal UI for large zoo animals appeared in 1991 and provided descriptions and ultrasonograms of the internal reproductive tract in Asian and African elephants, black rhinoceros, white rhinoceros, banteng, gaur, giraffe, and bactrian camel [126]. In elephants, ultrasonically detected changes in the cervix and uterus were described. Subsequent studies in Germany using transrectal UI have elucidated many of the unusual aspects of the reproductive cycle of the elephant that were unknown before UI became available [127]. The elephant was shown to be monovulatory with two follicular waves during the follicular phase. Recent studies have included color Doppler and three-dimensional UI to describe luteal and conceptus development in elephants [128]. In the original study [126], a nontranquilized rhinoceros was used to identify individual ovarian follicles for 34 days and an ultrasonogram of a 27-day embryo was recorded. The embryo of the rhinoceros resembled an equine embryo at a similar stage and was in a similar location at the caudal uterine horn. Ultrasound-guided oocyte recovery in the rhinoceros has also been reported [129]. The status of grayscale UI in the rhinoceros and elephant and the role of UI in assisted reproduction have been reviewed [130]. Researchers in Canada have used transrectal UI to study the ovaries in camels [131] and in several large species of wildlife; approximately 50% of the reports were in Theriogenology. The ovaries of elk (wapiti) were studied in detail for the anovulatory and ovulatory seasons [ ], leading to programs for ovarian synchronization, induction of follicular waves, and superovulation. Superstimulation of follicles, oocyte collection, embryo transfer, and ovarian synchronization programs have also been developed for bison after serial study of the ovaries by transrectal UI [135]. The reproductive organs of moose [136], muskoxen [137], and seals [138] were also studied by transrectal grayscale UI. 6. Specialized gray-scale UI techniques Ultrasound technologies have become increasingly important not only for viewing and characterizing the images of moving events (e.g., uterine contractions) and slow events (e.g., follicle growth), but also as a crucial component or guide for specialized techniques. The listing of the following techniques is intended as an overview, and preference is given for the reports that apparently introduced or popularized a technique: (1) first use of ultrasonically simulated biological structures (equine embryos) for reproduction research in any species [26]; (2) computerized pixel analyses of ovarian structures in mares [139,140], heifers[84 86,141,142], and ewes [143]; (3) observing images of semen streaming into the uterus as an aid for teaching and evaluating AI technicians [144]; (4) first attaching of echogenic markers in reproduction research in any species (horses [145]); (5) guiding cannulation into the caudal vena cava for sampling blood with a greater proportion of ovarian effluent in heifers [146]; (6) inserting a research substance directly into a follicle in mares [147] and heifers [148]; (7) sampling follicular fluid in heifers [149] and mares [150]; (8) sequential biopsy of the CL in heifers [151] and mares [152]; (9) aspiration of follicle contents for functional follicle ablation in mares [153] and heifers [154]; (10) recovering and transferring oocytes into other follicles by transvaginal UI in cattle [155] and mares [156] aided by manual transrectal manipulation of an ovary; (11) studying the developmental patterns of small follicles (1 3 mm) by transrectal UI in cattle [157]; (12) ultrasonic biomicroscopy for in vivo imaging of surface follicles as small as 0.4 mm (including the cumulus oocyte complex) in cattle, using an ultrasonic biomicroscope with a single crystal that emits

7 118 O.J. Ginther / Theriogenology 81 (2014) ultrasound waves of 20 to 70 MHz [158]; (13) evaluating moving structures such as the fetal heart by M-mode (motion mode) [2]; (14) transvaginal ultrasound guiding for inseminating and transferring embryos directly into a uterine horn by bypassing the cervix [159]; and (15) aspirating oocytes by transvaginal ultrasound scanning and guiding for in vitro fertilization [160]. Thus,theuseof specialized techniques further enhances the prestige of UI as a boon to research. On the applied side, the in vivo recovery of oocytes has become common in the embryo transfer industry subsequent to its publication in Theriogenology by researchers in The Netherlands [160,161]. 7. Color-Doppler UI and ultrasonography Fig. 7. Ultrasonograms from gray-scale UI and color Doppler UI of a longitudinal section of the spermatic cord of a bull. The gray-scale image shows anechoic sections of the highly convoluted testicular artery as it intertwines with the network of veins of the pampiniform plexus. Some of the arterial sections are rounded (arrows) with specular reflections on the upper and lower surfaces, indicating a smooth arterial wall and a perpendicular direction of the ultrasound beams. The color-doppler image shows arterial blood in red or blue depending on the direction of blood flow relative to the ultrasound beams. Arterial cross sections that are devoid of color (arrows) indicate that the ultrasound beams intersected the blood flow at a 90 angle so that the direction of flow was neither away nor toward the transducer. Adapted from [162] with permission. Doppler ultrasound is not used as frequently as grayscale ultrasound and therefore the principles of the Doppler technology are discussed briefly. The Doppler technology is based on Doppler-shift frequencies, wherein the frequency of echoes from moving red cells is increased or decreased as the cells move toward or away from the transducer [4,162,163]. The Doppler effect of ultrasound is similar to the Doppler effect of sound, wherein the sound frequency changes as the source (e.g., car horn) moves toward or away from a listener. Color-Doppler UI and Doppler ultrasonography provide distinctively different approaches for assessing the vascular system of reproductive organs. In color-flow mode (Doppler UI), the direction of blood flow relative to the face of the transducer is represented by different colors on the screen display (Fig. 7). The extent of color can be estimated by percentage of a tissue with color signals or can be calculated by computer, based on number of colored pixels. In spectral mode (Doppler ultrasonography), blood flow for a focused location in a specific artery is assessed by placing a sample-gate cursor (e.g., 1 mm wide) on the grayscale or color-mode image of the lumen of an artery (Fig. 8). The focused results from the sample gate are displayed on the screen by a graph that represents changing blood flow velocities at various times within a cardiac cycle or individual arterial pulse. The numerical velocities are computed and displayed on the screen for a selected cardiac cycle. Doppler indices (resistance index; pulsatility index) are also displayed on the screen. The indices are ratios computed from the various points of the changing velocities in the cardiac cycle. They are especially useful for the tortuous arteries of the reproductive tract because they are independent of the angle of the transducer to the angle of blood flow. These indices reflect the hemodynamics of the tissue supplied by the artery distal (downstream) to the sample gate. An increase in the resistance index or the pulsatility index indicates an increase in resistance and therefore a decrease in vascular perfusion of the tissues. Techniques for locating an image of a cross-section of the uterine and ovarian arteries in mares [162,164] and heifers/ cows [162,164,165] have been described. The locations of the pelvic arteries to the genitalia of stallions and bulls are also illustrated [162]. The use of transrectal Doppler ultrasonography for research studies in large animal reproduction was first reported by researchers in Germany in a series of publications in Theriogenology, beginning in 1998 in mares [164] and in 2000 in cows [165]. Ovarian and uterine blood flow velocities during the estrous cycle and uterine and umbilical blood flow during pregnancy were included. Their studies on blood velocity spectral graphs in cattle during the estrous cycle, pregnancy, and postpartum have been reviewed [163]. Vascular perfusion of the CL and uterus was greater during the first versus second follicular wave of the estrous cycle [166]. Color-Doppler displays of blood-flow signals in the follicle and CL in cattle have been popularized by researchers in Japan [ ]. The following research findings and conclusions are primarily from our laboratory and are used to illustrate the power of color Doppler UI and ultrasonography in reproduction research. Findings in mares include: (1) during follicle selection, blood flow in the follicle wall begins to increase in the future dominant follicle compared with the largest future subordinate follicle an equivalent of 1 day before diameter deviation [171] and is similar to the day of increasing prominence of the anechoic band [49]; (2) percentage of follicle circumference with blood flow signals begins to decrease in the follicle wall 4 hours before ovulation [172]; (3) serration of the granulosa opposite to the future site of ovulation results from blood vessels beneath the granulosa [173]; (4) blood flow in the wall of the dominant follicle (>30 mm) is less in anovulatory follicles than in ovulatory follicles during the transition between anovulatory and ovulatory seasons [174]; (5) percentage of wall of the preovulatory follicle with blood flow signals is greater in mares that subsequently become pregnant than for those that do not [175]; (6) percentage of the circumference of a preovulatory follicle with blood flow signals is greater and includes the apical area (site of potential rupture) when a hemorrhagic anovulatory follicle forms than when the follicle is ovulatory [176]; (7) neither an increase nor decrease in luteal blood flow was detected

8 O.J. Ginther / Theriogenology 81 (2014) Fig. 8. Doppler ultrasonogram of changes in blood flow velocity during an individual arterial or cardiac pulse. The sample gate (SG) delineates the small focus that will be the source of the graph of velocities in the selected cardiac cycle. The angle cursor (AC) is placed by operator to indicate the angle of blood flow to the plane of the ultrasound beams. Blood velocities of the systolic and diastolic portions of the cardiac cycle are depicted. Blood velocity is determined and listed on the scanner screen at various points of the velocity graph or spectrum. In this example, the average velocity is shown and is called time-averaged maximum velocity (TAMV). The resistance index is also shown. A greater index indicates less blood flow distal to the sample gate. Adapted from [162] with permission. before the beginning of a progesterone decrease in spontaneous luteolysis [177]; (8) after follicle evacuation, vascularization of the CL begins at the basal area (site of granulosa serration in the preovulatory follicle) and progressively extends toward the apical area during Days 0 to 6 [173]; (9) color-doppler signals apparently in the endometrium mark the future post-orientation site of the equine embryo proper well before the embryo proper is detectable by gray-scale imaging [178]; (10) heart rate of an equine embryo on Days 24 and 27 was lower by more than 3 standard deviations in an embryo that was lost on Day 31 than in controls [162]; (11) reduced uterine vascular perfusion is associated with uterine cysts [179]; and (12) the movement of the conceptus back and forth between uterine horns during embryo mobility is accompanied by a back-and-forth transient increase in vascular perfusion of the endometrium [63], and at the end of mobility perfusion is greater in the horn of fixation [63,180]. Color-Doppler UI research findings in heifers/cows include (1) the percentage of follicle wall with blood flow signals increases synchronously with the initiation of the LH surge [168]; (2) the percentage of wall of the preovulatory follicle with blood flow signals is greater in heifers that subsequently become pregnant [181]; (3) during luteolysis, blood flow in the CL increases during the ascending portion of each pulse of PGF2a metabolite, remains elevated for 2 hours and then decreases [182]; (4) the increased CL blood flow during PGF2a treatment is from the direct stimulation of PGF2a on the CL vasculature in heifers [183]; and (5) endometrial scores for an increase in the extent of vascular perfusion follow expansion of the allantochorion throughout each uterine horn in cattle [184]. These examples of research findings in mares and heifers highlight the capabilities of UI in reproduction research and would not have been possible without the availability of transrectal color- Doppler UI. A text and reference book with colored images and more detailed discussion has been published [162]. A study on vascular perfusion of the endometrium in pregnant mares is used to illustrate the approaches that can be used in uterine blood flow studies by the Doppler ultrasound technologies [63]. Vascular perfusion was assessed by the estimated score for number of color signals in a section of the endometrium, by computer-generated number of color pixels (color-doppler UI), and by blood velocity in various parts of an arterial pulse in an artery in the mesometrial attachment (Doppler ultrasonography). Each of the three approaches indicated similarity between the nonpregnant and pregnant mares until Day 12. On Day 13 of pregnancy, vascular perfusion by estimation of the percentage of color signals or by computerized pixel count was greater in the uterine horn with the mobile embryo. After fixation (end of embryo mobility), each of the two assessments by color-doppler UI and a lower pulsatility index in the assessment by ultrasonography indicated greater vascular perfusion in the horn of fixation than in the opposite horn. The heart beat of the equine embryo is detectable on Days 17 to 20 or approximately 2 days earlier by color- Doppler UI than for detection of the embryo proper by gray-scale UI [162]. Color-flow signals extend beyond the heart and into vessels of the membranes by Days 27 or 30. In cattle, color-flow signals extend into the vessels by Day 28 and are detectable in the umbilical cord, aorta, and carotid artery. Color Doppler images of the embryo (before Day 40) and fetus (after Day 40) are shown for horses and cattle (Fig. 9). Extensive series of color-doppler images for the follicles, CL, uterus, embryo, and fetus in horses and cattle and examples of determination of blood velocities and resistance indices by ultrasonography have been published [162]. 8. Handling of research data Despite the more than 30-year history of ultrasonic technologies as research tools, there seems to be a reluctance to apply statistical analyses to data obtained by grayscale or color-doppler UI, except for data from studies of follicles and follicular waves. Many other quantitative end points are available through the imaging technologies [2,3,6] as indicated by the examples in previous sections.

9 120 O.J. Ginther / Theriogenology 81 (2014) Fig. 9. Color-Doppler ultrasonograms of the embryonic vesicle and fetus in a mare and in a heifer. as, allantoic sac; ao, aorta; ca, carotid artery; dv, ductus venosus; ep, embryo proper; fl, front limb; ht, heart; hv, hepatic vein; hl, hind limb; jv, jugular vein; mm, mesometrial attachment; uma, umbilical artery; umv, umbilical vein; vc, vena cava; ys, yolk sac. Adapted from [162] with permission. End points that can be quantified and compared among groups include: (1) diameter or other dimensions of the embryonic vesicle, embryo proper, and fetus and other measurable structures involving the conceptus; (2) diameter, area, or volume of the CL; (3) embryo and fetal heart rate; (4) extent of embryo mobility measured by distance and time in mares; (5) diameter or other measures of the uterus (Fig. 10); (6) height or other measures of fluid pockets in the uterus; and (7) percentage of a structure (e.g., CL) with color pixels, indicating the extent of vascular perfusion. With regard to follicles, diameter is a convenient measure that is readily understood. However, there may be instances when surface area (as calculated from a cursor tracing of the periphery) may be more meaningful because function is most related to the periphery of the antrum [2]. Irregular structures (e.g., equine CL) can be traced for area (cm 2 ) determination. Many scanners have convenient capabilities for obtaining such measurements and displaying the numerical results on the screen. Thus, ultrasound quantitative data can be used for statistical comparisons among time or groups similar, for example, to data for changes in hormone concentrations. Scoring of end points from minimal to maximal (e.g., 1 4) is a useful technique for image characteristics that are not amenable to quantifying by conventional measuring techniques (Fig. 10) [2]. Scoring can be useful, for example, for relative amounts that cannot be easily measured, such as intrauterine fluid, endometrial edema, extent of uterine contractions, changes in uterine shape, and percentage estimate of a defined area with color signals. The scoring approach facilitates profiling and statistically analyzing changes and analyzing temporal relationships or correlations among the ultrasonically monitored events as well as among other events, such as changes in hormone concentrations. The scoring system is subjective in that the operator s judgment is used. Bias can be minimized by filming the images so that scoring can be done without knowledge of source, or scoring can be done by a second Fig. 10. Examples of a quantitative measurement (height from dorsal to ventral surface) and a subjective score (0 [nil] to 4 [maximum]) for a bovine uterus. Each end point can be subjected to statistical analyses for differences among days. Adapted from [6] with permission.

10 O.J. Ginther / Theriogenology 81 (2014) operator who is unaware of the source or even the hypothesis under test. We have developed considerable confidence in the scoring approach in our laboratory because of consistent results among experiments and independent operators and the ease of analyzing and interpreting results in relationship to known biological mechanisms. Information from gray-scale and color-doppler UI can be digitized and analyzed by computer. Digitization is a procedure in which each pixel of an image is assigned a numerical value for brightness in gray-scale UI, or the number of colored pixels is counted in color-doppler UI [2].Conversion of image information to numerical data allows the use of quantitative statistics for temporal characterizations or testing of hypotheses. However, the sophisticated processing does not eliminate bias. The operator retains the responsibility of selecting and delineating the areas to be processed, and these decisions are notoriously subject to bias. Computer pixel analyses, therefore, can be dangerous and lull the operator into a false sense of confidence. It is best if the operator selecting the images or delineating the area for study does not know the experimental group or source of the image. Pixel analyses have been used, for example, to characterize day-to-day changes in the intensity or echogenicity of the CL [86,140], quantify changes in echotexture [85] and shape changes in the preovulatory follicle as ovulation approaches [139], and evaluate the extent of vascular perfusion [4,162]. Detailed information on statistical handling of ultrasonic images and pixel analyses in cattle [6] and horses [3] is available. The images displayed on the ultrasound screen represent interactions between the technology and living tissues [2]. Artifactual and factual echoes are produced, and consequently, embarrassing errors may be published. Proper instrument adjustment and proper interpretation of the echoes on the ultrasound screen are crucial. Interpretation requires knowledge of the relationships between tissues and echoes and the ability to differentiate between true and artifactual responses. There are two types of grayscale reflections (specular and nonspecular) that are presented as echoes [2]. Artifactual echoes can be mistaken for echoes that represent actual structures. In addition to specular echoes, certain tissue formations also cause ultrasonic waves to bend (refract), bounce back and forth or re-echo (reverberate), become weakened (attenuated), entirely blocked (shadowing), or exaggerated (enhanced). As a result, distortions appear on the ultrasonic image, which can be mistaken for normal or pathologic structures or changes. Artifacts are especially common during UI of the reproductive tract because of the many pockets of bowel gas, fluid-filled structures, and the pelvic bone. Artifacts are a nuisance in color-doppler work [162]. Clutter artifacts for Doppler UI and distorted graphs of the cardiac cycle in Doppler ultrasonography are most common in transrectal examinations and result from movements of tissues, the animal, or transducer. Animal restraint or acclimation and filter settings can be used to lessen the clutter. In this regard, detomidine sedation in horses and xylazine sedation in heifers affects blood flow in major arteries (e.g., internal iliac) but an effect on local vascular perfusion in the ovaries and endometrium has not been detected [185]. 9. The male Stallions and bulls have not been neglected in the utilization of transrectal and transcutaneous gray-scale and color Doppler UI [3,6,162]. Transrectal gray-scale UI apparently was first used in stallions in 1987 in a description of the accessory sex glands [186]. Researchers in Idaho described the changes in the accessory sex glands before and after sexual preparation (teasing of mares) [187]. The vesicular glands became rounded after sexual preparation from the entry of vesicular gel, as indicated by an increased anechoic area. The Idaho researchers also developed a unique system for transrectal UI of the sex glands during ejaculation [188,189]. Apparently for the first time in any species, images of the dynamics of the sex glands were observed in real time. Rhythmic pulses (emissions) of the prostate secretions occurred before the onset of the manually determined penile and urethral rhythmic contractions or sequential ejaculatory pulses. Discharge of the prostate continued during the first few ejaculatory pulses. Rhythmic ampullary and prostatic activity began a few seconds before the start of ejaculation. Ampullary fluid passed through the ducts in distinct boluses before ejaculation, and prostate emission continued for the first few ejaculatory contractions. In contrast, the emission of vesicular glands with accompanying pulses in the ducts occurred after ejaculatory or urethral contractions were underway (Fig. 1). These ultrasonically determined patterns of emission are consistent with the order of appearance of the various portions of the seminal fluid in stallion ejaculates. The results demonstrated the power of UI in physically active organs. The technique for transcutaneous imaging of the external genitalia in stallions has been described [190]. A standoff pad is used on the ventral surface of the testes for imaging the branches of the testicular artery. The central vein can be imaged running longitudinally. Abnormalities (e.g., tumors, focal inflammation) can be imaged. Abnormalities of the epididymis and the cause of scrotal enlargement (e.g., gravitated fluid, inguinal hernia, blood clot) also may be detected by transcutaneous UI. The genitals of bulls have also been examined by grayscale UI. The examinations are effective for assessment of structure and for generating research data [6,191]. Reports on the initial imaging of the bull testis in vitro in 1987 [192] and in vivo in 1988 [193] and the ultrasonic appearance of the accessory sex glands [191] were first published in Theriogenology. Mean pixel intensities from ultrasonograms of testes parenchyma indicate that gray-scale UI may be a useful noninvasive method for determining reproductive development in maturing bulls [194]. The accessory sex glands are imaged transrectally and the testes, epididymides, and the testicular cones, transcutaneously. The vascular cone consists of the highly coiled testicular artery (Fig. 7) intertwining with the venous network the pampiniform plexus. Recent reports concluded with the opinion that the primary use of UI in the assessment of reproductive function in bulls is the detection of lesions in the testes and scrotum [195], but assessing breeding soundness is limited [196]. Recent reviews are available for the many publications on monitoring reproductive function by UI in bulls [196,197].