Research applications of ultrasonic imaging in reproductive biology.

Research Applications of Ultrasonic Imaging in Reproductive Biology1f2 P. G. Griffin and 0. J. Ginther Department of Veterinary Science, University of Wisconsin-Madison 53706 ~

ABSTRACT: In the short time that transrectal ultrasonic imaging of the reproductive tract has been used as a research tool, many discoveries have resulted, some of which would otherwise have escaped detection for many years. Ultrasonography provides a wide array of morphologic information without invading or disturbing the tissues. Examinations can be done repeatedly over many days, or a dynamic event (e.g., ovulation) can be monitored in its entirety by continuous observation (e.g., 30 min). Inclusion of ultrasonic examinations in experimental protocols affords the opportunity to associate changing morphology with hormonal and other functional

changes. If experimental testing is expected to involve changing morphology, ultrasonic imaging should be considered. End points can be measured or ranked and therefore data can be statistically analyzed for conventional hypothesis testing. The research potential of this technology and its adaptability for computer-assisted assessment go f a r beyond simplistic determination of ovulation, luteal formation, and pregnancy diagnosis. It is the authors' opinion that ultrasonic imaging is a revolutionary advance in reproductive research that is destined to rival the impact of other technologic breakthroughs, including radioimmunoassay.

Key Words: Ultrasonography, Reproduction, Research

J. Anim. Sci. 1992. 70:953-972

Introduction Prior to the introduction of real-time transrectal ultrasonic imaging in large animal reproductive research, the morphology of the reproductive organs was evaluated by transrectal palpation, at surgery, or after excision. The dynamic aspects of morphology were largely inaccessible. Real-time ultrasonic imaging has provided a noninvasive and nondismptive technique to image directly, in situ, the internal and external anatomy of reproductive organs and tissues, and to characterize reproductive events (e.g., ovulation, transition of the uterus from a diestrous to an estrous echotexture). The technique allows frequent or continuous (e.g., for 30 min) evaluation of individual animals,

'Supported by the College of Agric. and Life Sci., Univ. of Wisconsin, Madison. Resented at a symposium titled "Applic& tion of Ultrasound in Animal Science Research" at the ASAS 82nd Annu Mtg., Ames, IA. %e authors thank L. Kulick and R. Gneiser for technical assistance, M. Westphal for manuscript preparation, and G. P. Adams for valuable comments on the manuscript. Received November 5, 1990. Accepted March 22, 1991.

enabling the study of sequential relationships within and among structures. Some clinical and research applications of large animal ultrasonic imaging of the reproductive tract have been reviewed in the last several years (equine: Ginther, 1986, 1988a; McKinnon et al., 1987a,b; Squires et al., 1988; Fontijne and Hennis, 1989; bovine: Kastelic et al., 1988; Pierson and Ginther, 1988; Kahn and Leidl, 1989; caprine and ovine: Buckrell, 1988). However, the use of this technology as a reproductive research tool has not been emphasized. The purpose of this report is to review current and potential applications of ultrasonic imaging in reproductive research, especially in the large farm animal species. Examples will be given of ranking and quantitative approaches that are amenable to statistical analysis.

Ultrasonic Principles and Techniques Detailed reviews of the basic principles and transrectal techniques of ultrasonic imaging in large animals are available (Ginther and Pierson, 1983; Ginther, 1986; Pierson et al., 1988; Ligtvoet et al., 1989).The following brief synopsis of ultrasonic

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fundamentals is drawn from these sources. Ultrasonic imaging utilizes high-frequency sound waves to produce images of the tissues and internal organs. The sound waves are produced by vibrations of specialized crystals (piezoelectric crystals) housed in the ultrasound transducer. Vibrations of the crystals are produced by pulses of electric current. The sound waves sample a wide (e.g., 50 d, but thin (e.g., 2 d, tissue area, resulting in a two-dimensional image, representative of a thin slice of tissue analogous to a histologic section. The sound waves are directed through the tissues of interest by moving and varying the angle of the transducer. The ultrasonic characteristics of a tissue depend on its ability to reflect sound waves. The proportion of sound waves reflected back to the transducer is converted to electric current and displayed as an echo on the ultrasound viewing screen. The transducer, therefore, acts as both the sender of sound waves and the receiver of echoes. The echoes are evident on the viewing screen as varying shades of gray (black to white). Liquids do not reflect sound waves Ke., are nonechogenic or anechoic) and are represented on the viewing screen as black (or white, depending on the scanner format). The ultrasonic images of liquidcontaining portions of structures such as ovarian follicles and embryonic vesicles appear black. Dense tissues (e.g., bone, diestrous cervix) reflect a large proportion of the transmitted sound waves Le., are echogenic) and are represented on the viewing screen as light gray or white. The various soft tissues and contents of the reproductive tract appear on the screen in varying shades of gray, depending upon their echogenicity. The pattern of the interspersed shades of gray provides an echotexture that may be characteristic of a given tissue during a given reproductive state. Most ultrasound scanners used today in reproductive research are B-mode (brightness modality), real-time scanners. In B-mode ultrasonography, the image is a two-dimensional display of dots (pixels);the brightness of the dots is proportional to the amplitude of the reflected echoes returning to the transducer. Real-time refers to the ability to image movement (e.g., fetal motion or heartbeat) as it occurs. Videotape recording of real-time ultrasound examinations is an effective tool for studying the dynamics of reproductive structures and for detailed study of discrete reproductive events that entail motion (e.g., ovulation, embryo mobility, uterine contractions). Available ultrasound scanners vary widely in capabilities and incidental provisions, such as portability, transducer and cable design, annotation and measuring provisions, freeze frame memory, zoom and magnification, and photographic and videotape capabilities. Image quality varies with the number of crystals or elements in the Downloaded from https://academic.oup.com/jas/article-abstract/70/3/953/4632139 by guest on 01 August 2018

transducer, frame rate (number of images/second), focusing methods, and many other aspects of design. A scanner should provide images that are not marred by a checkered or pixelated appearance. Some scanners that are currently marketed are of poor quality and cannot be considered suitable for research purposes. For a detailed discussion of the selection of ultrasound scanners, see Ginther (1986). Transrectal scanning is the most common approach to ultrasonic examination of the reproductive tract in large animals. Because of the close proximity of the reproductive tract to the rectal wall, transrectal scanning allows the use of highfrequency transducers, resulting in detailed images. Transducers of varying frequencies are available and are selected on the basis of size and location of the structures of interest. The most commonly used frequencies in large animal reproduction are 3.5, 5.0, and 7.5 MHz. The higher the frequency of the transmitted sound waves, the better the image resolution, and the shallower the depth of penetration. Therefore, relatively small structures (e.g., a 4-mm ovarian follicle) located close to the transducer are well suited for study with a 5.0- or 7.5-MHz transducer. In contrast, large structures located relatively far from the transducer (e.g., mid- to late-gestation fetus and uterus) are best suited for study with a 3.5-MHz transducer, because depth of penetration is more important than a detailed image. It is emphasized that frequency (MHz) is an indicator of resolution, but not of image quality. Preparation and precautions for transrectal scanning are similar to those for transrectal palpation. Especially for research purposes, examining conditions should be optimized (e.g., scanner screen at eye level, subdued external light). A coupling medium (e.g., carboxymethylcellulose; Ginther, 1986) is used as both a lubricant and to provide contact or elimination of air between the rectal wall and the transducer. Air reflects almost 1000!0of ultrasound waves and, therefore, prevents the entry of waves into the tissues. Transrectal scanning is most commonly used in mares (review, Ginther, 1986) and cows (review, Pierson and Ginther, 19881, used increasingly in llamas (Adams et al., 1989b, 1990, 1991~1,and has been reported for sows (Cartee et al., 1985; Thayer et al., 19851, ewes [Buckrell et al., 1986; Buckrell, 1988; Gearhart et al., 19881,goats (Dorn et al., 19891,deer [Bingham et al., 1990), giraffes (Gilbert et al., 19881, and various large, nondomestic species CAdams et al., 199lb). In those animals too small to be examined with a handheld intrarectal transducer, stiffening of the transducer’s coaxial cable (e.g., with a heavy piece of hose) to form an extension allows external intrarectal manipulation of the transducer. The use of an extension in the larger

ULTRASOUND IN REPRODUCTIVE RESEARCH

species (e.g.,mare, cow) may alleviate much of the animal straining commonly associated with prolonged (e.g., 30 mid transrectal examination. Extensions have also been used for transrectal imaging of the most cranial portions of the reproductive tract of very large species (e.g., elephant; Adams et al., 199lb)and for transvaginal imaging in cattle (Pieterse et al., 1990bl. The transabdominal approach (e.g., through the inguinal region) has been used in many species h a r e : Pipers and Adams-Brendemuehl, 1984; Adams-Brendemuehl and Pipers, 1987; sow Inaba et al., 1983; Cartee et al., 1985; Thayer et al., 1985; Botero et al., 1986; ewe: Haibel, 1986, 1988; Kelly et al., 1987; Gearhart et al., 1988; Haibel and Perkins, 1989; goat: Haibel, 1986; deer Mulley et al., 1987) and is particularly appropriate for animals or species thought to be too small for transrectal examination. Hair, wool, and excessive subcutaneous fat are obstacles to transabdominal imaging. Imaging of the ovaries via a transvaginal approach has been reported in cows Pieterse et al., 1988, 1990b; Pieterse, 1989) and is used extensively in women. The transvaginal approach in cattle involved simultaneous transrectal positioning of the organs. Artifacts are common during imaging of the reproductive tract because of the presence of many fluid-filled structures (e.g., embryonic vesicle, ovarian follicle) and close proximity to gasfilled loops of bowel and the bony pelvis. Air introduced into the abdominal cavity during laparotomy can interfere with ultrasonic examination of the tract for several days after surgery and effort should be made to expel air prior to closure (Pierson et al., 1988). Sound wave artifacts include angled reflections [bending), reverberation (reechoing), attenuation (weakening), shadowing (blockage), and enhancement or through-transmission these artifacts are discussed in detail elsewhere (Ginther, 1986). Artifactual echoes complicate the interpretation process and can be mistaken for normal or pathologic structures or changes. Interpretation of ultrasonic images becomes increasingly sophisticated with experience. Many structures and changes that may be unapparent to a beginner may be obvious to an experienced examiner. Even after much experience, however, a structure may be noted for the first time and thereafter be a source of wonderment as to why it was overlooked for so long. Scientists who remain alert and receptive during scanning may be

3For example, IMAGE-l/AT Image hocessing and Analysis System, Universal Imaging Corporation, Media, PA. 4For example, Polaroid Bravo Computer Slide Maker, Polaroid Corporation, Cambridge, MA.


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rewarded with new, often exciting, observations. As computer technology advances, many sophisticated digital image processing and analysis systems are becoming available that can be directly applied to reproductive research using ultrasonic imaging3. Initial computer processing involves the digitalization of stored (e.g., on videotape) ultrasonic image information. The digitized information can then be quantified and(or1refined for subsequent analysis and review. Numerous programs are available, including applications for image enhancement (e.g., image averaging, image sharpening, edge or border enhancement), quantitative measurements (e.g., point-to-point distance, cross-sectional area, perimeter, shape analysis), gray-scale expansion, brightness analysis (e.g., pixel gray-scale distribution), and image storage and retrieval. Applications are also available that can process and reproduce digitized, stored images (e.g., on floppy diskl directly onto photographic film for presentation, publication, or hardcopy storage*.

Ultrasonic Imaging of the Ovaries Follicles The ultrasonic anatomy of the ovaries of the mare (Ginther and Pierson, 1984a,b; Ginther, 1988a), cow Bierson and Ginther, 1984a Edmonson et al., 1986; Pieterse, 19891, and llama Udams et al., 1989b) has been described in detail. There WEU close agreement between assessment of ovarian structures by transrectal ultrasonic imaging and by slicing of excised ovaries for mares (Ginther and Pierson, 1984a) and heifers Bierson and Ginther, 1987e). Similarly, morphologic characteristics of excised mare ovaries ultrasonically imaged in a water bath closely mirrored the gross characteristics of the same ovaries sliced in a plane approximating the plane of the ultrasound images (Ginther and Pierson, 1984a). On the viewing screen, ovarian follicles appear as black tnonechogenic), roughly circumscribed areas that are spherical to irregular in shape. Irregular shapes are attributable to compression between adjacent follicles or between a follicle and luteal structures or ovarian stroma. Follicles as small as 2 to 3 mm are identifiable with a high-quality scanner and high-frequency transducer; however, counting small follicles can be laborious and, depending on the hypothesis under test, may not be needed. The reliability of transrectal ultrasonic imaging for counting and measuring follicles in heifers was assessed by comparing, in the blind, results of in vivo ultrasound examinations and slicing of ovaries within 4 h of ultrasound examination (n = 42

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ovaries; Pierson and Ginther, 1987e). Correlation coefficients between ultrasonography and slicing ranged from .80 to .92 for various follicle diameter categories (2 to 3 mm, 2 2 mm, 2 4 mm, 1 7 111111, 2 11 mm). With ultrasonography, there was a tendency to slightly overestimate the number of 2- to 3-mm follicles per ovary (ultrasonography, 16.4 f .7;slicing, 15.5 f .81 and 2 2-mm follicles (ultrasonography, 21.8 f .9; slicing, 20.8 f .41. The numbers of ultrasonically detected follicles in these diameter categories were well within the range of follicle numbers detected in similar diameter categories in an extensive necropsy study (Rajakoski, 1960). These results indicated that transrectal ultrasonic imaging is a reliable method for identifying and measuring follicles as small as 2 to 3 mm. A recent report stated that "it was impossible to count the follicles 5 to 10 mm in diameter accurately, and only 34.3% of these follicles were detected (Pieterse et al., 199ob). However, these workers may have used a scanner of inferior quality, despite its 5.0-MHz rating. One approach to the study of folliculogenesis involves ultrasonically monitoring populations of follicles in various categories according to antral diameter. With this approach, the relationships among follicular populations have been characterized over time during the estrous cycle in mares (Ginther and Pierson, 1984a; Pierson and Ginther, 1987a) and heifers Bierson and Ginther, 1984a, 1987c,8 and during early pregnancy in mares (Ginther, 1986; Ginther and Bergfelt, 1990) and heifers, Bierson and Ginther, 1986, 1987b3. This approach has been used in heifers to test hypotheses regarding effects of right and left sides and Downloaded from https://academic.oup.com/jas/article-abstract/70/3/953/4632139 by guest on 01 August 2018

intraovarian relationships (Pierson and Ginther, 1987b,c,dl. Ultrasonic monitoring of individually identified follicles has recently been found to be a powerful tool for studying follicular dynamics during the estrous cycle and pregnancy, and the intraovarian relationships among dominant and subordinate follicles and the corpus luteum (Figure 1). In this approach, the location and diameter of individual follicles is indicated on a n ovarian sketch, using landmarks that include the cranial and caudal ovarian poles, the greater curvature of the ovary, and the relative position among follicles and luteal structures. Identifications can be done at the time of scanning or later from videotapes, but provisions should be made to minimize bias. Investigators seem to agree that it is not practical to identify and monitor individual follicles until they reach 4 to 5 mm in cattle and 10 mm in horses, because of the large number of smaller follicles. Using this approach in heifers, a predominance of two (Pierson and Ginther, 1988; Ginther et al., 1989b,c; Knopf et al., 19891 or three (Savio et al., 1988; Sirois and Fortune, 1988)follicular waves per estrous cycle have been found. In heifers, monitoring individual follicles has been used to test hypotheses regarding the intraovarian and systemic roles of the dominant follicle during the estrous cycle (Ginther et al., l989al and pregnancy (Ginther et al., 19898, the effect of prostaglandin Fza on follicular dynamics and selection and development of the ovulatory follicle (Quirk et al., 1986; Kastelic et al., 1990b1, the temporal associations between FSH surges and the initiation of a follicular wave (Adams et al., 1991a1, and the effects of exogenous progesterone (Sirois and Fortune, 1990; Bergfelt et al., 19911, injections of follicular fluid mastelic et al., 199Oc; Turzillo and Fortune, 19901, and cauterization of the dominant follicle (KOet al., 1991) on follicular wave patterns. In mares, this approach has been used to study folliculogenesis during the estrous cycle (Sirois et al., 1989) and spring transitional period (Ginther, 1990).

Ultrasonic imaging has also been used in cows to assess follicular status before the initiation of superovulatory treatment and to predict and monitor the ovarian response to such treatment (Guilbault et al., 1988); numbers and sizes of follicles and the timing of ovulations were monitored. Follicular activity and luteal status in cows under progestin treatment have also been monitored ultrasonically (Jones et al., 1989). Transabdominal (flank approach; Calleson et al., 1987) and transvaginal ultrasonic imaging (Pieterse et al., 1988)have been used to facilitate follicular aspiration of bovine oocytes. Ultrasonic imaging clearly offers tremendous advantages over alternative

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approaches (transrectal palpation, excision of ovaries, marking individual follicles) for the study of follicular dynamics. Recently, the ultrasonic morphology and dynamics of the ovaries have been described for the llama [Adams et al., 1989b, 19901. Follicular populations and wave patterns were characterized and the effects of lactation and reproductive status (anovulatory, ovulatory nonpregnant, pregnant) on follicular dynamics were elucidated. The nature of the llama as an induced ovulator and its suitability for transrectal scanning make the species an interesting model in which to study folliculogenesis and ovulation. Little published information was found involving ultrasonic assessment of the ovaries in sheep and goats. In a brief communication, it was reported that transrectal ultrasonic imaging (7.5MHz transducer) was used to monitor follicular activity and luteal status in superstimulated goats (Dorn et al., 1989). This apparent success of transrectal imaging of ovaries in goats indicates that a similar approach may be feasible in sheep. Stiffening of the coaxial cable (e.g., with hose) should allow external manipulation of the intrarectal transducer in these small species. The ultrasonic morphology of normal ovaries in sows has apparently not been described. One study reported the transabdominal imaging of ovarian follicular cysts in three of six sows (Botero et al., 1980). In a limited trial in our laboratory, it was observed that transrectal scanning with a handheld transducer was feasible in swine. The ovaries, however, were not consistently located and because of the length and orientation of the uterine horns, systematic examination of the entire tract was difficult. However, these problems may be obviated with experience, and it is our impression that transrectal scanning of the sow’s reproductive tract is a potentially fruitful research tool. Transabdominal ultrasonic imaging has been used to monitor follicular growth in rhesus monkeys and to evaluate the ovarian response to exogenous gonadotropins (Morgan et al., 198713). Similarly, transrectal scanning was used to monitor ovarian activity in cyclic giraffes in response to hormonal manipulation (Gilbert et al., 1988). The ultrasonic characteristics of the preovulatory follicle in the mare have been studied extensively. Follicle shape changed from spherical to nonspherical in 84% of 79 preovulatory periods and follicle wall thickness increased as the interval to ovulation decreased (Pierson and Ginther, 1985a). Similarly, in another study (Carnevale et al., 19881, flattened or irregular images of preovulatory follicles were noted as ovulation approached in association with reduced follicular tone. RecentDownloaded from https://academic.oup.com/jas/article-abstract/70/3/953/4632139 by guest on 01 August 2018

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Figure 2. Effect of shape of preovulatory follicle on distributions of lime lengths. Line lengths were determined by pixel analysis and represented the distances from the center pixel of the follicle to each pixel at the perimeter of the follicle. (A) Circular cross-sectional follicular image 24 h before ovulation. (B) Semicircular follicular image 2 h and 17 min before ovulation. (C) Oblong follicular image 23 min before ovulation. Note the changes in the spread of line length distribution as follicles changed shape from circular to oblong. (Adapted from Townson and Gmther, 1989a.)

ly, ultrasonic evaluation of diameter and shape changes of the preovulatory follicle has been refined with the use of digital image analysis (Townson and Ginther, 1989a). With this technique, ultrasonic image information is digitalized and analyzed by computer. Digitalization is a procedure in which each pixel of an image (collectively, pixels form the ultrasonic image) is assigned a numeric value for brightness and location coordinates. The pixel information may then be used objectively for statistical analyses. This approach has been termed pixel analysis and was used to assess follicle size and shape by calculating line lengths within each follicle for the distance between the center pixel of each follicle and each pixel forming the follicle perimeter. Mean and median line length were used to determine follicle size and variance in line length was used to evaluate follicle shape (Figure 21. The disappearance of a previously detected preovulatory-sized follicle is an indication of recent ovulation. In mares, the ovulation site can be visualized on the day of ovulation even without knowledge that a large follicle was present on the previous day. In a trial conducted to test the ability to detect an ovulation site (Ginther, 1988a1, the ovulatory ovary was correctly identified, in the blind, in all of 24 mares that had a single ovulation and were at d 0 (day of ovulation). All 21 mares that had not ovulated were also correctly identified, except for one that was recorded as uncer-

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tain. In addition, subsequent formation and identification of a corpus luteum confirms ovulation. In the mare, the extent and pattern of follicular fluid release and the fate of the discharged fluid at the time of ovulation have been studied in detail (Townson and Ginther, 1987, 1989b).Frequent and, at times, continuous observations were used. Two patterns of follicular evacuation were observed during continuous observation: 1) an abrupt loss of follicular fluid in which a substantial majority of the fluid I> 90%) disappeared within 1 min, and 2) a gradual loss of follicular fluid in which approximately 50% of the fluid disappeared within 1 min. Averaged over both patterns, the mean time for disappearance of 80% of the follicular fluid was 90 s. Apparent rents or breaks in the follicular wall toward the ovulation fossa (Carnevale et al., 1988) and apparent extraovarian fluid collections in the area of the ovulation fossa (Townson and Ginther, 1989b3 were detected in association with follicular evacuation. The collections were continuous with the fluid of the evacuating follicle and disappeared at about the same time or shortly after the end of evacuation. This observation supported the hypothesis that follicular fluid does not collect in the ipsilateral oviduct and illustrates how the technology has opened a difficult research area. As a further example of the utility of ultrasonic imaging, dramatic shape changes in adjacent nonovulatory follicles during evacuation of the ovulatory follicle have been used as an indicator of changing intraovarian pressure (Townson and Ginther, 1989a). In cows, the process of ovulation has apparently not been studied with continuous scanning. However, in a study of 10 cows examined by ultrasonography every 2 h, it was noted that the preovulatory follicle changed shape from oval to elongated before ovulation (Rajamahendran et al., 1989). Ultrasonic examinations in heifers and cows at 2-h (Rajamahendran et al., 1989) and 4-h (Larsson, 19871 intervals have been used to detect the timing of ovulation for determining relationships among estrus, ovulation, hormonal levels, and body temperature.

Corpora Lutea The ultrasonic characteristics of the corpus luteum and luteal dynamics have been studied in mares, heifers, and llamas. In a study of 55 interovulatory intervals in mares (Pierson and Ginther, 1985131, the corpus luteum was ultrasonically detectable with a 5.0-MHz transducer for a mean of 17 d; the mean interovulatory interval was 22 d. In heifers, the corpus luteum was first detectable on d 0 (73Y01, d 1 (l6%), and d 2 to 4 (1l%l, and was thereafter identifiable to approximately 1 d after the next ovulation Eastelic et al., 199Od).A Downloaded from https://academic.oup.com/jas/article-abstract/70/3/953/4632139 by guest on 01 August 2018

well-defined border to the luteal gland was visible after approximately d 3. A comparison of ultrasound and postslaughter detection of the corpus luteum on d 12 and 14 was made in the blind (Pierson and Ginther, 1987e). There was 100% agreement between the two techniques for identification of the corpus luteum-bearing ovary and presence of a cavity within the gland. Recently, Pieterse et al. (1990b) stated that the sensitivity (ability to detect all corpora lutea) for mid-cycle corpora lutea in cattle was only 83.3 and 80.6% for transrectal palpation and ultrasonic imaging, respectively; both techniques were inaccurate for detection of young and old corpora lutea. As for the follicular work (noted above), however, these seemingly discouraging findings are attributable to the use of a scanner of apparently inferior quality. Two distinct luteal morphologies have been described in the mare Bierson and Ginther, 1985b). Approximately 50% of corpora lutea were uniformly echogenic (central nonechogenic cavity < 10%of luteal cross-sectional area) throughout their period of detectability; the remaining 50% developed a centrally located, nonechogenic area representative of a blood clot. In corpora lutea that formed a central clot, the average time of first detection of the clot was 30 h after follicular evacuation; the size of the central area gradually increased and reached maximum at 44 to 128 h (Townson and Ginther, 1988). Subsequently, its size decreased over the life of the gland Bierson and Ginther, I985b). No differences between the two types of glands in plasma progesterone concentration or ultrasonically determined luteal tissue area were detected (Townson and Ginther, 1989c,d). These data, obtained with sequential ultrasonic examination, have elucidated an aspect of luteal development in the mare that had escaped appreciation before the availability of ultrasonic imaging; the data do not support the well-ingrained notion (Ginther, 1979) of a consistent formation of a corpus hemorrhagicum before the development of a mature luteal gland. Digital image analysis Ke., pixel analysis), as previously described, was also used to assess the echogenicity of luteal glands during the equine estrous cycle (Townson and Ginther, 1989d). Each pixel forming the image of the luteal gland received a computerized gray-scale value of 1 to 256 (minimal to maximal echogenicity). Mean gray scale values were then generated for each luteal image. Additionally, a subjective scoring system (scored 1 to 8; minimal to maximal echogenicity) was used to assess the same images (Figure 3). Results obtained with the two assessment techniques closely paralleled one another. Echogenicity was greatest during the 24 to 48 h after the

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onset of luteal development and has been hypothesized to be reflective of luteal hemodynamics (Ginther, 1986). The pixel analysis approach illustrates how quantitative and objective data can be obtained with ultrasonic imaging. The results also demonstrated, however, that image assessment can be done by visual scoring techniques when provisions are made to preclude bias in the assignment of scores. Two types of luteal morphology have also been described in the cow, corpora lutea with and without a fluid-filled central cavity. The central cavities appear ultrasonically as nonechogenic (black) to hypoechogenic areas surrounded by relatively hyperechogenic luteal tissue Bierson and Ginther, 1987e, Figure 4). Central cavities of varying sizes 12 to > 10 mm in diameter) occurred in 79% (63/801 of luteal glands in one study (Kastelic et al., 1990dl. Ultrasonically determined luteal tissue area (Kastelic et al., 199Od), plasma progesterone concentrations (Kit0 et d.,1986; Kastelic et al., 1990al, and pregnancy rate (Kastelic et al., 199Odl were not affected by the presence or size of a central cavity, and it was concluded that the central cavities were not functionally important. The entire subject of cystic ovarian structures is amenable to reinvestigation because of the ability to monitor the origin and life of fluid-filled cavities by ultrasonic imaging. Plasma progesterone concentration and ultrasonically determined luteal tissue area and diameter in heifers were highly correlated during the estrous cycle (Sprecher et al., 1989; Kastelic et al., 1990al. Similarly, there was a positive correlation between plasma progesterone concentration and luteal diameter in nonpregnant and pregnant llamas [Adam et al., 1991~).In studies of luteal dynamics in mares (Bergfelt et al., 1989) and heifers [Kastelic et al., lQQOa), changes in plasma progesterone concentration closely paralleled observed changes in the ultrasonic cross-sectional area of the corpus luteum. In pregnant mares, a resurgence in growth (cross-sectional areal of the primary corpus luteum was discovered in association with increasing progesterone concentrations and the release of equine chorionic gonadotropin (Bergfelt et al., 1989). In nonpregnant heifers and llamas, ultrasonically detected regression of the corpus luteum began approximately 2 d later than functional regression (Kastelic et al., 1990a; Adams et al., 1991~).The close relationships between luteal size and progesterone concentration in these species indicates that ultrasonic assessment of the corpus luteum is a valuable adjunct to plasma progesterone assay as an index of luteal function (Kastelic et al., 1990a; Figure 1). It should be noted, however, that under unusual conditions (e.g.,heifers fed endophyte-infested hay; Patterson Downloaded from https://academic.oup.com/jas/article-abstract/70/3/953/4632139 by guest on 01 August 2018

et al., 19891, luteal-like structures may be detected even though plasma progesterone concentrations are e 1 ng/mL.

Ultrasonic Imaging of the Uterus The dynamic morphology of the uterus has been studied by ultrasonic imaging in the mare, cow, and llama. However, ultrasonic studies on uterine morphology and dynamics in sheep, goats, and sows are lacking. The report of Pierson and Ginther (lQ87fJillustrates that many detailed and quantified or scored uterine end points are accessible by ultrasonic imaging and can be statistically analyzed (e.g., amounts of vaginal and uterine fluid and uterine size, shape, and echotex ture; Figures 5 and 6). Uterine ultrasonic echotexture (characteristic image) provides, in effect, an instant indicator of the prevailing hormonal status (estrogen or progesterone dominance). In both

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Figure 3. Comparison of changes in luteal echogenicity in mares using a subjective scoring system (1to 8; minimal to maximal echogenicity) vs pixel analysis. There was an effect of time ( P < .05) for both techniques. The two techniques closely paralleled one another in estimation of luteal echogenicity over time. Differences between sequential stars or plus signs are significant ( P < .05). (Adapted from Townson and Ginther, 1989d.)

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Figure 4. Ultrasonograms of bovine corpora lutea with (A) no detectable central cavity, (B]a s m a l l central cavity, and (C) a large central cavity. Boundaries of corpora lutea are indicated by arrows. The scale on the left margin is in centimeters. (Adapted from Kastelic et al., 1990d. Reprinted with permission from Butterworth-Heineman).

cows (Pierson and Ginther, 19878 and llamas CAdams et al., 1989b1, uterine echotexture w a characteristically dark during the period corresponding to follicular dominance (estrus),reflecting an extensive degree of endometrial edema. Ultrasonic echotexture changes in the mare are primarily the result of changes in the degree of edema of the longitudinal endometrial folds (Ginther and Pierson, 1984c; Hayes et al., 1985; review, Ginther, 1986; Figure 7). During estrus, there is marked edematous expansion of the endometrial folds. Estrous echotexture is characterized by alternating and intertwining areas of hyper- and hypoechogenicity; the hyperechogenic areas (white) are attributable to the dense, connective tissue core of the folds and the hypoechogenic (dark) areas are attributable to the edematous outer portion of the folds. Diestrus echotexture is marked by minimal discernible endometrial folding, resulting in a relatively homogeneous image. In a study of the seasonal effects on uterine ultrasonic morphology (Hayes et al., 19851, ultrasonic echotexture was scored based on the degree of endometrial folding (1 to 3;minimal to maximal). Unexpected increases in echotexture score occurred on d 4 and 5 during May and June, but not during September and October, suggesting that a surge of estrogen occurred during diestrus early in the ovulatory season. Such surges have not been identified with conventional hormonal assay. This observation illustrates how the technology can give direction to future studies. In mares, the pattern of change in uterine echotexture during the estrous cycle closely paralleled the pattern of change in the intensity of estrous behavior (Hayes et al., 19851. Estrous behavior is attributable to estrogen exposure (Ginther, 19791, and this relationship indicates that ultrasonic characteristics of the uterus provide a Downloaded from https://academic.oup.com/jas/article-abstract/70/3/953/4632139 by guest on 01 August 2018

sensitive indication of estrogen exposure (Hayes et al., 1985). In the mare, partitioning of the estrous cycle into follicular and luteal phases, based on the ultrasonic echotexture of the uterus and the detectability of the corpus luteum, was an accurate tool for estimation of the stage of the estrous cycle (Ginther and Pierson, 1989). In 98% of follicular phases, a uterine echotexture characteristic of estrus or intermediate between estrus and diestrus was detected on at least 1 d in the 3 d preceding ovulation. These authors speculated that ultrasonic partitioning of the estrous cycle into estrus and diestrus may approximate the accuracy of hormonal partitioning (estrogen and progesterone concentrations) and may exceed the accuracy of behavioral partitioning. Transrectal ultrasonic imaging has permitted assessment of changes in shape of the uterus during various reproductive states in heifers Bierson and Ginther, 1987f; Figure 61, and llamas (Adam et al., 1989b). The in situ findings are in sharp contrast to the artifactual impressions resulting from transrectal palpation and from examination of excised reproductive tracts illustrating, again, the research power of ultrasonic imaging. Under luteal dominance, the uterine horns of cows and llamas were maximally curled. During follicular dominance, the uterine horns were less curled in cattle and nearly straight in llamas. Thus, in situ imaging of bovine uterine horns indicates decreased curling during estrus, contrasting with the impression from transrectal palpation of increased curling (BonDurant, 1986). Similarly, in llamas, the ultrasonic images of ventrally and caudally curled uterine horns contrasts with the short, blunt, and straight horns of camelids seen at necropsy (Mobarak and El Wishy, 1971). Pronounced changes in the shape of the uterine horns durirm the estrous cycle in cows and llamas


probably reflect, at least in part, the degree of interstitial pressure associated with edema. Dynamic changes in uterine size during the estrous cycle have been characterized in mares and cows using transrectal ultrasonic imaging. In cows, both the vertical diameter of the uterine body and the distance from the dorsal surface of the uterus to the uterine lumen were greatest during the period associated with estrus Bierson and Ginther, 19878. In mares, hyperechogenic

U t o r i n o Fluid

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markers, surgically attached to the uterine serosa, were used to sequentially measure the diameter of uterine horns at specific sites (Griffin and Ginther, 1991b); the diameter of the horns was greatest during estrus and smallest during mid- to latediestrus. Uterine diameter in mares was positively correlated with scores for endometrial echotexture (Figure 8). In cows, maximal uterine diameter occurred during estrus, when uterine tone is maximally turgid (Roberts, 1986). Maximal diameter also occurred during estrus in mares, but, in contrast, uterine tone is minimal (flaccid) during estrus. This species difference may indicate that the myometrium in mares has a greater capacity to relax an6 contract in response to hormonal fluctuations than does that in cows. That is, in mares, as the endometrium becomes edematous and expands during estrus, the myometrium relaxes to accommodate the expansion, resulting in greater uterine diameter and flaccid uterine tone. This may explain the enigma, in mares, of a circumscribed ultrasonic appearance of crosssections of uterine horns during estrus (Ginther, 19861, whereas, by transrectal palpation, the uterus is perceived as thin-walled and flaccid. In cows, the myometrium may not relax to the same degree as in mares, resulting in myometrial resistance to edematous endometrial expansion and a substantial increase in interstitial pressure, which is expressed as turgid uterine tone and an uncurling of the uterine horns during estrus. This

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Figure 5. Number of uterine horn cross-sections in one 5-MHz field of view and scores for intrauterine fluid, uterine shape and ultrasonographic echotexture during the interovulatory interval (n 58) in heifers. There was an effect of day (P < .0001)for each end point. Intrauterine fluid scores, 0 to 3 (none detectable to maxima! accumulation); uterine shape scores, 1 to 4 (minimal to maximal curling, see Figure 6);echotexture scores, 1 to 3 (representative of a diestrus, intermediate, and estrous uterus). The number of uterine horn crosssections in one 5-MHz field is an objective indication of the degree of curling of the horns. (Adapted from Pierson and Ginther, 1987f.)

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Figure 6. Diagrammatic representation of the shape of bovine uterine horns based on ultrasonic observations. Numbers represent coded values for scoring uterine shape. A score of 1 was characteristic of estrus; a score of 4 was characteristic of diestrus. (Adapted from Pierson and Ginther, 1987f.)

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example illustrates the utility of ultrasonic imaging for developing hypotheses regarding physiologic processes based on sequential observations and demonstrates how ultrasonic and palpation data can be assimilated into unifying hypotheses across species. Transabdominal ultrasonic imaging has been used in rhesus monkeys to assess cyclic morphologic changes in the uterus during the menstrual cycle (Morgan et al., 1987a). As in the mare, cow, and llama, the follicular phase was characterized by a relatively less echogenic endometrium, presumably due to edema, and by an increase in uterine diameter. Transrectal ultrasonic imaging has been used to noninvasively monitor uterine contractile activity in mares. Contractile activity was assessed from 1-min scans of the uterine body. Each scan was assigned an overall uterine activity score based on the extent of to-and-fro movements of endometrial tissue reflectors and myometrial waves or indentations that moved along the ventral uterine surface. In two separate studies conducted by independent operators (Cross and Ginther, 1988; Griffin and

Ginther, 19901, maximal uterine activity in nonpregnant mares occurred on d 14 to 18, corresponding temporally with the reported period of luteolysis and associated prostaglandin Fza release (Ginther, 1079; Figure 9). In pregnant animals, maximal activity occurred 4 d earlier than in nonpregnant mares, during the reported period of maximal embryo mobility (Leith and Ginther, 1984); uterine contractions are a primary impetus for embryo mobility (discussed in next section). Based on close agreement between results obtained by independent operators in the blind and findings that were readily interpretable in terms of known physiologic events, it was concluded that ultrasonography is a valid and repeatable technique for quantifying uterine activity (Griffin and Ginther, 1990). Thus, ultrasonic imaging provides a tool for immediate sequential assessment of uterine contractility without invading the uterus or attaching or inserting monitoring devices (e.g., electrodes). This technique has also been used to assess the role of ovarian steroids on uterine activity using seasonally anovulatory

Figure 7. Ultrasonogram of the equine uterus during estrus and diestrus. A, B, D, and E are longitudinal sections of the uterine body; C and P are cross-sections of uterine horns. In image D, the upper and lower limits of the uterine body are indicated by arrows. The arrow in image E indicates the uterine lumen, identifiable as a hyperechogenic (white) line when viewed longitudinally. (Adapted from Ginther, 1986.) Downloaded from https://academic.oup.com/jas/article-abstract/70/3/953/4632139 by guest on 01 August 2018

963

ULTRASOUND IN REPRODUCTIVE RESEARCH Nonbred mares

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Figure 8. Uterine horn diameters and endometrial echotexture scores for nonbred mares. Diameters were measured at mid-horn, at a specific site identified with a surgically attached marker. Echotexture scores are on a scale of 1 to 4 based on the degree of endometrial folding (minimal to maximal folding). Horn diameter .75; and endometrial score were highly correlated (r P < .0001). (Adapted from Griffin and Ginther, 1991b).

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mares (Cross and Ginther, 1987).Similar ultrasonic uterine activity studies apparently have not been reported in other species. The ultrasonic morphology of the equine postpartum uterus has been described (Ginther and Pierson, 1984~)and uterine involution in the cow (Okano and Tomizuka, 19871 and mare CMcKinnon et al., 1988) has been investigated by ultrasonic imaging. In cows, uterine involution was complete at approximately 40 d based on ultrasonic assessment of uterine horn diameters. In the mare, based on ultrasonic measurements of the uterine horns, involution was complete in a mean of 23 d; the previously gravid horn was recognized as the larger horn for a mean of 21 d. In this same study, ultrasonography was used to evaluate the effects of progestin therapy on pregnancy rates, uterine size, intrauterine fluid collections, and the rate of involution. Small (e.g.,height on the ultrasound screen of 5 to 10 mml collections of free intrauterine fluid in mares under progesterone dominance were discovered (Ginther and Pierson, 1984~)by ultrasonic imaging and were believed (Ginther et al., 1985) and later c o n f i i e d (Adams et al., 1987) to be an indication of an inflammatory process. Intrauterine fluid collections are ultrasonically visible as mobile, free, nonechogenic (fluid-fiied) areas with poorly defined borders within the uterine lumen. In a herd of research ponies, mares with a history of embryonic loss on d 11 to 15 and mares with a history of small intrauterine fluid collections during diestrus had several similarities: 1) reduced pregnancy rates, 21 reduced progesterone concentrations at d 7 and 11, 3) shortened interovulatory Downloaded from https://academic.oup.com/jas/article-abstract/70/3/953/4632139 by guest on 01 August 2018

intervals for both the intervals associated with embryo loss and the interval in which an embryo was not detected, and 41 repeatability of the conditions within individual mares (Ginther et al., 1985; Ginther, 1986). In the subsequent study (Adams et al., 19871, intrauterine fluid collections during diestrus were associated with endometrial inflammation (determined by endometrial biopsy), premature luteolysis and an abbreviated interovulatory interval, decreased pregnancy rate at d 11, and an increased embryonic loss rate between d 11 and 20. These small fluid collections would likely not be detected except by ultrasonography and, in effect, provide an immediate indication of endometritis. In a subsequent study (Adams and Ginther, 1989a1, ultrasonic detection of intrauterine fluid collections was used to identify mares with endometritis and to determine the efficacy of intraute&e infusion of blood plasma as a treatment. Uterine cysts are ultrasonically visible as immobile, usually compartmentalized, nonechogenic structures with well-defined borders (Ginther and Pierson, 1984~1.Mares with a large number and(or1 size of cysts tended to have a reduced pregnancy rate at d 40 CPldams et al., 1987) and, in another ultrasound study (Chevalier-Clement, 19891, the embryonic loss rate (mean, c d 441 was increased in mares with cysts compared with unaffected mares. However, because cysts were more common in older mares (Adams et al., 19871, the relationship between cysts and fertility may be confounded by age. Ultrasonic imaging has pro-

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Figure 9. Uterine contractile activity scores for pregnant and nonpregnant pony mares as determined by ultrasonography. Activity scoring was on a basis of 0 to 4 (minimalto maximal activity). There was an effect of day (P < .01) and a day x reproductive status (pregnant vs nonpregnant) interaction (P < .01). (Adapted from Cross and Ginther, 1988.)

964

GRIFPIN AND GINTHER

vided a much needed means for continuing assessment of uterine cysts without interfering with their development. Fetal remnants (e.g., fetal bones, m&ied fetuses) are ultrasonically detectable as brightly echogenic structures within the uterine lumen (Ginther and Pierson, 1984~1.Many smaller bone remnants (e.g., 6 I11112) are difficult to detect with transrectal palpation, and their identification and removal is greatly facilitated by ultrasonography. The ultrasonic appearance of some bovine uterine abnormalities has been described Pissore et al., 1986; K&hn and Leidl, 19891. Clearly, ultrasonic imaging is an effective tool for diagnosing and studying uterine pathology and clarifying its relationship to fertility and for evaluating treatment regimens. In the cow, transrectal ultrasonic imaging has also been used to train artificial inseminators

I........................,

Figure 10. Diagrammatic representation of the equine uterus and intrauterine mobility of the early embryonic vesicle. The uterus has been divided into nine segments. Shown are the sequential locations of a d-14 conceptus determined every 5 min over a 2-h period. The numbers are the number of minutes the vesicle spent in each segment and the arrows indicate the direction of vesicle movement. (Adapted from Ginther, 1984b. Reprinted with permission from Butterworth-Heinemann). Downloaded from https://academic.oup.com/jas/article-abstract/70/3/953/4632139 by guest on 01 August 2018

meal et al., 19891; a retrievable echogenic bead was deposited in the uterine lumen and its location determined. The potential for similar applications in reproductive research remains largely untapped.

Ultrasonic Imaging of the Conceptus Another application of ultrasonic imaging in reproductive research involves the detection and study of the conceptus. Real-time scanning has provided a means to visualize directly the products of conception and their development and viability. In the mare, the embryonic vesicle can fiist be detected on d 9 or 10 with a high-quality scanner (Ginther, 1984a). The embryo proper can be imaged by d 20 or 21 and its heartbeat can be visualized by d 22 (Ginther, 1984a, 1986). The following aspects of equine embryonic development have been discovered, clarified, or confirmed by ultrasonic imaging: 1) in situ growth profiles of the embryonic vesicle and the embryo proper, 2) shape changes of the vesicle during early pregnancy, 31 regression of the yolk sac and related expansion of the allantoic sac, and 4) ascent and descent of the embryo proper within the vesicle and the associated development of the umbilical cord keview, Ginther, 1986). Additionally, by means of ultrasonic imaging, the phenomena of equine embryo mobility (Ginther, 1983a; Figure 101, fixation, and orientation (Ginther, 1983b) were discovered and hypotheses regarding their nature were tested (Ginther, 1984b, 1985% Leith and Ginther, 1985; Kastelic et al., 1987; Bessent et al., 1988). The effects of various factors on the extent and nature of embryo mobility were studied by developing a system of mobility trials (one vesicle location determination every 5 min for 2 h); thus, data that could be analyzed were obtained. Simulated embryonic vesicles (fluid-filled rubber objects) have been used to study the mechanisms involved in embryo mobility (Ginther, 1985a1, and the conceptus has been monitored after ligation of segments of the uterus (McDowell et al., 1985). The use of simulated vesicles illustrates how research protocols can be developed to enhance the utility of scanners. Ultrasonic imaging has been used extensively in the past few years for the characterization of the nature of twinning in mares (reviews, Ginther, 1986, 1989a,b). Investigations into twinning involved clarifying the origin of twins (Ginther, 1987; Pascoe et al. 1987; Ginther and Bergfelt, 19881, characterizing the growth and development of twin embryos (Ginther, 1984d, 19871, discovering the embryo-uterine interactions involved in the twin embryo phenomenon (Ginther, 1984d, 1987,


1989~1,and calculating the probability and timing of embryo reduction (natural elimination of one member of a twin set) under specific conditions (relative size and location of the two embryos; Ginther, 1984c, 1988b, 1989~).The twin embryo problem had defied meaningful study until ultrasound scanners became available. Ultrasound research was the sole technology used in the development of the deprivation hypothesis for the mechanism of embryo reduction (Ginther, 1989~). Equine fetal development from 60 to 335 d of pregnancy has been studied with transrectal ultrasonic imaging ( K W and Leidl, 19871. In horse mares, using a 5.0-MHz transducer, fetuses were visualized 75% of the time and growth profiles for the fetal eye socket, cranium, ribs, stomach, and trunk were developed. Ultrasonography has been used to characterize fetal activity and mobility within the allantoic cavity and dynamic shifts in the distribution of allantoic fluid within the uterine lumen during early pregnancy (Figure 11; Griffin and Ginther, 1991a).Transabdominal scanning has also been used to image the late-gestation equine fetus, and it was suggested that imaging of the fetus may be useful for prenatal evaluations (Pipers and Adams-Brendemuehl, 1984; AdamsBrendemuehl and Pipers, 1987).

965

The bovine conceptus has been studied in detail with ultrasonic imaging, and there are numerous reports on the use of ultrasonography for pregnancy determination (Pierson and Ginther, 1984b; Taverne et al., 1985; Chaffaux et al., 1986; Boyd et al., 1988; Wilson and Zalesky, 1988; Hughes and Davies, 1989; Kastelic et al., 1989a, 1991a Pieterse et al., 199Oal. Presumptive early diagnosis (prior to detection of an embryo proper) is based on the detection of a discrete, nonechogenic structure or line (height of 1.5 to 2.0 I11I11) within the uterine lumen but must be subsequently confirmed by the progressive elongation of the nonechogenic area and eventual detection of the embryo proper. Definitive diagnosis was unreliable before d 18 using a 5.0-hfHZ transducer (Kastelic et al., 1989a) and before d 16 with a 7.5-MHZ transducer (Kastelic et al.,1991a). Early diagnosis based solely on the detection of fluid within the uterine lumen was unreliable for two reasons: 11 the small dimensions of the elongating conceptus approach the limits of resolution of most available scanners and 2) small amounts of apparently normal free intrauterine fluid are present as early as d 10 and can be indistinguishable from the vesicle Kastelic et al., 1991al. The embryonic heartbeat is detectable on d 21 or 22 (Pierson and Ginther, 198413;

F'igure 11. Ultrasonograms of a uterine horn segment from one mare on d 77 of pregnancy. A, B, and C are crosssectional images of the mid-portion of the middle cornual segment. A steel bead (large arrows] was attached to the uterine serosa and serves to indicate that the three images are of the same site. A and B were taken within 1 min of each other and C was taken 22 min later. Small arrows indicate the periphery of the uterine horn. Nonechogenic areas (black)indicate the changing amount of allantoic fluid at this marked site. (Adapted from Griffii and Ginther, 1991a). Downloaded from https://academic.oup.com/jas/article-abstract/70/3/953/4632139 by guest on 01 August 2018

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Curran et al., 1986a). Elongation and height of the embryonic vesicle, development and growth rate of the embryo proper, and the day of fist detection of various embryonic and extraembryonic structures and fetal movement have been elucidated (Curran, 1986a,b).Ultrasonic morphology, growth profiles, and position and orientation of the fetus throughout the fetal stage have been described (K&hn, 1989, 19901; the fetus was consistently imaged during the f i s t 7 mo of gestation; thereafter, approximately one-third of examinations failed to reach the fetus. A recent application of ultrasonic imaging in fetal studies is the development of a highly accurate technique for the ultrasonic diagnosis of fetal sex in mares and cows. Sex diagnosis in cows between 73 and 120 d can be based on observation of the scrotum and mammary glands (Miiller and Wittkowski, 19861. Earlier diagnosis can be based on the relative location of the genital tubercle (forerunner of the penis and clitoris). The optimal interval for sex diagnosis based on position of the genital tubercle in mares was 59 to 68 d (Curran and Ginther, 1989); in cows, reliable diagnoses began on d 55 and extended through the last day of the experiment (d 00) (Curran et al., 1989). Correct diagnoses in cows were made on d 50 to 110, according to a short communication (Wideman et al., 1989). In swine, transabdominal and transrectal ultrasonic imaging have been used for pregnancy determination. In one study (Cartee et al., 19851, the earliest day of pregnancy detection with transrectal scanning was 21 d postbreeding. The fetal heartbeat was visible at 25 d postbreeding and fetal body movements were detected at 28 d. In another study (Thayer et al., 19851, the mean day of first detection of the conceptus was 15 d by transrectal scanning (5.0-MHz transducer) and 30 d by transabdominal scanning (3.5-MHz transducer). The results of these studies indicate that ultrasonic imaging is a potentially valuable tool to study the early events of pregnancy and the development of the conceptus in swine. Ultrasonic imaging has been used in sheep and goats primarily as a means for pregnancy diagnosis and estimation of fetal numbers (review, Buckrell, 1988). In one study (Gearhart et al., 19881, the earliest that pregnancy was correctly diagnosed with transrectal scanning was 20 d postbreeding. In the same study, the earliest identification of multiple fetuses, transrectally, was 3 1 d postbreeding; the probability of correctly determining fetal numbers was approximately 80% with both transrectal and transabdominal approaches between d 25 and 50. No reports of systematic studies on the ultrasonically monitored development of the conceptus in sheep or goats were found. TransabdomiDownloaded from https://academic.oup.com/jas/article-abstract/70/3/953/4632139 by guest on 01 August 2018

nal ultrasonic imaging has been used to assess biparietal diameter of sheep and goat fetuses between approximately d 40 and 100 (Haibel, 1988; Haibel and Perkins, 19891. These measurements were reliably predictive of gestational age during the second trimester. Transabdominal scanning was also used to study the relationships between ultrasonic measurements of the head and thorax and fetal weight (Kleeman et al., 1987). Measurement of thoracic diameter was highly correlated with fetal weight at slaughter. These results illustrate the potential for ultrasonic monitoring of fetal growth and development, in situ. Placentomes are ultrasonically visible by 35 to 40 d in small ruminants using a transabdominal approach (Haibel, 19801 and by d 35 in heifers using transrectal scanning (Curran et al., 1986b). They are visible as C-shaped or circumscribed areas of echogenicity adjacent to or within the placental fluid. In heifers, the placentomes increased in size until the end of the experiment (d 60) and appeared larger in the area of the embryo or fetus. The ultrasonic diameter of placentomes in ewes has been studied from 45 to 141 d; maximum diameter of the structures was reached on d 80 (Kelly et al., 1987). The diameter and postmortem weight of placentomes were highly correlated on d 94 and 141; it was concluded that ultrasonic imaging is potentially valuable for the study of placentome development and placental growth and viability in the ewe and other ruminants.

Ultrasonic Imaging of Embryonic Loss Timing of death of the embryo is an important aspect of studies characterizing embryonic loss, and precise in situ timing was not possible before the availability of ultrasonic imaging. Ultrasonically detecting the loss of a heartbeat allows the use of the same criterion for determining time of death of an embryo as for the death of an adult; precision is limited only by the frequency of examination. This ability, alone, gives credibility to the statement that ultrasonic scanners are tremendous research tools. The ability to detect and monitor the equine embryo from a very early stage (d 9 or 10) has enabled critical study of early embryonic loss (review, Ginther, 19861. Ability to monitor the embryonic vesicle beginning on d 9 to 11 is fortuitous, because these days precede the interval for embryo blockage of the uterine luteolytic mechanism. The ultrasonic morphology of spontaneous and induced embryonic loss in mares has been described (Ginther, 1985b; Ginther et al., 1985). Early losses (before detection of an embryo proper) generally occurred without ultrasonic indi-


cations of impending loss (Ginther et al., 1985). However, some early losses were presaged by the development of small collections of ultrasonically detectable intrauterine fluid outside the embryonic vesicle. Ultrasonic indicators of loss at later stages included a failure of fixation or dislodgment after fixation, absence of a heartbeat, an echogenic ring or mass floating in a collection of fluid, an echogenic area in the dead embryo, and an apparent gradual decrease in the amount of placental fluid with disorganization of placental membranes. Ultrasonic imaging has also been used as a tool to evaluate the effects of time of insemination relative to time of ovulation on pregnancy rate and embryonic loss rate in mares (Woods et al., 1990). Spontaneous (Chaffaux et al., 1986; Kastelic et al., 199lb) and induced (Kastelic and Ginther, 1989b) embryonic loss in cattle has been studied with ultrasonic imaging. In one study Mastelic et al., IgQlb),the association between time of spontaneous embryonic death (lack of heartbeat) and luteal regression [based on ultrasonically measured luteal areal, and the fate of the conceptus after embryonic death were examined. In spontaneous losses between d 20 and 40 in which luteal regression preceded embryonic death, the conceptus was lost quickly with minimal degeneration. When embryonic death preceded luteal regression, there was ultrasonic evidence of gradual conceptus degeneration; conceptus fluid and its breakdown products were maintained for prolonged periods. In all losses, based on failure to ultrasonically detect conceptus remnants and on ultrasonic detection of cervical patency, the conceptus was apparently eliminated by expulsion through the cervix (Kastelic and Ginther, 198913; Kastelic et al., 199lb), thereby throwing into question the dogma of embryo resorption.

Ultrasonic Imagin in Wild and Endangerecf Species The applications of real-time ultrasonic imaging in the study of reproductive physiology in wild or endangered species is in its inception. The feasibility of detecting changes in morphology of the tubular genitalia and detecting and measuring ovarian structures by transrectal ultrasonic imaging in several large, nondomestic species was recently demonstrated (Adams et al., 199lb).Ultrasonic images of the reproductive tract were obtained in the rhinoceros, elephant, banteng, gaur, and giraffe. In one white rhinoceros, individual ovarian follicles were identified and monitored over a 34-d observational period, and in one black rhinoceros, an early conceptus (27 d postbreeding) was imaged, including detection of an embryo Downloaded from https://academic.oup.com/jas/article-abstract/70/3/953/4632139 by guest on 01 August 2018

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proper with a heartbeat. Additionally, pregnancy was detected in a banteng cow (48 d),a giraffe (13 mol, and a bactrian camel (3.5 mol. In the banteng, impending embryo loss was suspected based on failure to detect a heartbeat in the embryo proper. It was concluded that ultrasonic imaging provides a needed research tool for the elucidation of certain aspects of reproductive biology in these endangered species. Transabdominal ultrmonic imaging was used to diagnose pregnancy and assess fetal growth in four captive bottlenose dolphins Williamson et al., 1990).Pregnancy was c o n f i i e d from the 4th mo of gestation by detection of placental fluids and fetal movement. Viability of fetuses was assessed based on observations of fetal heart rate and body movement. Growth curves for the diameter of the skull and thorax were also developed. Transrectal (Bingham et al., 1990) and transabdominal (Mulley et al., 1987) approaches have been used to determine pregnancy in deer. Reliable diagnoses with transrectal scanning were possible from 40 to 50 d and the embryo was evident in some animals by d 30. Profies for uterine, amnion, and placentome diameter, crown-rump length, and thoracic diameter have been developed ultrasonically for the red deer (Bingham et al., 1990). Transrectal scanning was also used to monitor follicular development and the response to an ovulation induction protocol in a Przewalskii’s horse mare Durrant and Hoge, 1988).Clearly, ultrasound offers tremendous potential for the elucidation of reproductive morphology and events in endangered species.

Ultrasonic Imaging

. of the Male Reproductive System Trsnscutaneous ultrasonic imaging has been used to characterize the ultrasonic morphology of the testicles of boars (Cartee et al., 1986) and bulls Bechman and Eilts, 1987; Eilts and Pechman, 1988). Ultrasonic measurement of testicles was correlated with testicular circumference, weight, and volume but not with scrotal circumference in bulls (Cartee et al., 19891 and with daily sperm output in stallions (Love et al., 1990). Ultrasonic scanning of the bovine testicle was found to have no effect on reproductive capacity (semen characteristics, testicular consistency and dimensions) after exposure to a 3-min scan with a 5.0-MHz transducer (Coulter and Bailey, 1988). Establishment of normal ultrasonic parameters for testicular dimensions and characterization of normal ultrasonic morphology are necessary to enable investigators to study degenerative and disease processes of the testicle. This technique is directly applicable to other species.

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The ultrasonic anatomy of the accessory sex glands of the bull Weber et al., 19881 and stallion (Little and Woods, 1987) has been described. Transrectal techniques were used in both studies to measure the vesicular glands, ampullae, and bulbourethral glands, and the prostate, seminal colliculus, masculine uterus, and deferent ducts were also studied in the stallion. In bulls, ultrasonic measurements were found to be repeatable in individual animals over time and, in both species, in was concluded that the anatomic relationships and organ dimensions were accurately imaged by ultrasonography. Changes in the morphology and dimensions of the accessory sex glands before and after sexual preparation and after ejaculation were studied in the stallion by transrectal scanning (Weber et al., 1990). An exciting aspect of these ultrasonic studies involves the ability to visualize the pulsatile discharge of the ejaculate in real-time. These studies have laid the basis for future investigation into the nature of the ejaculatory process and specific accessory gland function during ejaculation.

Conclusions In the short time that ultrasonic imaging has been used as a research tool, many discoveries have resulted, some of which would otherwise have escaped detection for many years. Included among these discoveries are the following: 1) the phenomena of embryo mobility and fixation in mares, 21 the mechanism of natural embryo reduction in mares, 3) the natural uncurling of the uterine horns during estrus in heifers and llamas, 4) the incidental, rather than obligatory, formation of the corpus hemorrhagicum in mares, 5 ) the differential patterns of uterine contractility in mares during the estrous cycle and corresponding days of pregnancy, 6 ) the patterns of follicular evacuation in mares, 7) the precise nature of growth and regression of individual follicles in heifers, mares, and llamas, 81 resurgence in growth of the primary corpus luteum in association with the release of equine chorionic gonadotropin, and 9) expulsion of conceptus debris through the cervix after embryonic death in cattle and horses rather than elimination by resorption. Maximal utilization of ultrasonography provides a wide array of morphologic information but requires good equipment and examining conditions, keen observational skills, and creative utilization. The use of simulated structures (e.g., water-filled balloon for the equine embryonic vesicle), marking a specific area (e.g., suturing a marker to the uterus), and guiding and locating an inserted device (e.g., insemination pipette, biopsy Downloaded from https://academic.oup.com/jas/article-abstract/70/3/953/4632139 by guest on 01 August 2018

needle) illustrates how the power of ultrasonography can be even further extended. End points can be used that are subject to measurement or ranking and therefore can be analyzed for convential hypothesis testing. The research potential of this technology and its adaptability for computerassisted assessment go f a r beyond simplistic determination of ovulation, luteal formation, and pregnancy diagnosis. The ultrasound technique has been touted as a revolutionary research tool in reproductive biology (Ginther, 1980). Despite this high expectation, the technique has not been well integrated into experimental protocols. For example, only one ultrasonogram had appeared in the Journal of Animal Science as of July 1990 (Cross and Ginther, 1988). The first reports on the use of transrectal ultrasonography in the cow appeared in 1984. However, examination of issues of the Journal of Animal Science from 1985 to July 1990 disclosed 111 reported in vivo studies of reproductive biology of the cow, and none of these used ultrasonography-not even to define a standard reference point (e.g., day of ovulation) or end point (e.g., pregnancy). In our opinion, at least 81% (90/111) of these studies would have been considerably improved by transrectal ultrasonic imaging. Inclusion of ultrasonic examinations in the experimental protocols would have afforded the opportunity to associate changing morphology with hormonal and other functional changes. If the experimental testing is expected to involve a change in morphology (and it usually does),ultrasonic imaging should be considered. We continue to believe that transrectal ultrasonic imaging is a revolutionary advance in reproductive biology research that is destined to rival the impact of other technologic breakthroughs, including radioimmunoassay. Furthermore, the technology adds a measure of excitement to research because of the ability to observe dynamic reproductive events as they occur, including events that were previously unknown.

Implications Ultrasonic imaging offers reproductive biologists a noninvasive and nondisruptive technique for directly evaluating reproductive organs and tissues. As such, it provides the opportunity for sequential study of individual structures with the potential to relate changing morphology to function. Reproductive events that were previously unobservable, and often unknown, can now be imaged as they occur. Quantitative approaches that are amenable to statistical analysis, including computerized image analysis, can be used for


conventional hypothesis testing. The potential of ultrasonic imaging as a research tool in reproduction cannot be approached, however, unless keen observational skills are developed and creativity is maximized.

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