ilable at ScienceDirect

Theriogenology 81 (2014) 112–125

Contents lists ava

Theriogenology

journal homepage: www.theriojournal .com

40th Anniversary Special Issue

How ultrasound technologies have expanded and revolutionized researchin reproduction in large animals

O.J. Ginther a,b,*a Eutheria Foundation, Cross Plains, Wisconsin, USAbDepartment of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA

a r t i c l e i n f o

Article history:Received 25 June 2013Received in revised form 6 September 2013Accepted 7 September 2013

Keywords:Arterial pulseColor-Doppler imagingDoppler ultrasonographyGray-scale imagingVascular perfusion

* Corresponding author. Tel.: þ1 608 798 3777; faE-mail address: ginther@vetmed.wisc.edu.

0093-691X/$ – see front matter � 2014 Elsevier Inchttp://dx.doi.org/10.1016/j.theriogenology.2013.09.0

Gray-scale ultrasonic imaging (UI) was introduced in 1980 and initially was used toexamine 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 hasbeen used extensively to characterize follicle dynamics and investigate the gonadotropiccontrol and hormonal role of the follicles. However, the use of transrectal UI has alsodisclosed and characterized many other aspects of reproduction in each species, including(1) endometrial echotexture as a biological indicator of circulating estradiol concentra-tions, (2) relative location of the genital tubercle for fetal gender diagnosis by Days 50 to60, 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 theuterus on Days 11 to 15, (2) how one embryo of a twin set eliminates the other withoutself-inflicted damage, and (3) serration of the granulosum of the preovulatory follicleopposite to the future rupture site as an indicator of imminent ovulation. Studies withcolor-Doppler UI have shown that vascular perfusion of the endometrium follows theequine embryo back and forth between uterine horns and follows the expansion of thebovine allantochorion throughout each horn. In heifers, blood flow in the CL increasesduring the ascending portion of an individual pulse of PGF2a metabolite and then de-creases. These examples highlight the power of UI in reproduction research. Without UI, itis likely that these and many other findings would still be unknown.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

“Iwish I could seewhat’s goingon in there,”was a lamentof farm animal reproductive biologists and cliniciansthroughout most of the 20th century. During the 1980s,gray-scale (B-mode) ultrasonic imaging (UI) of the repro-ductive organs in horses and cattle revealed dynamic yetunexpected events (e.g., intrauterine travels of the equineembryo, acrobatic feats of the equine and bovine fetus).Before the availability of UI, such activities were not on theresearcher’smenuor couldnot be studiedwithoutworryingabout invasive interference. The physical nature of knownevents could be contemplated but not described (e.g.,

x: þ1 608 798 3722.

. All rights reserved.07

timeline and characteristics of follicle evacuation duringovulation).

This reviewdiscusses the early history and eventual loftystatus of ultrasound technologies in reproduction research.Mares and, a few years later, heifers/cows provided theincentive for the initial adaptation and enthusiasm for gray-scale UI. The premier event (as in Columbus discovering theNew World) was a report in Theriogenology in 1980 byPalmer and Driancourt [1] on UI of the reproductive organsof the mare. The report was published a century after thediscovery of the piezoelectric effect of certain crystals [2].Crystals in ultrasound transducers have the piezoelectriccharacteristic of expanding and contracting in response toalternating polarities of electric signals. The crystals in thetransducer produce ultrasound waves when applied to tis-sue and receive and convert the resulting echoes back into

O.J. Ginther / Theriogenology 81 (2014) 112–125 113

electric signals. The signals are converted and displayed asshades of gray that represent the intensity of the echoes andthe locations of the tissue reflectors. Sounds simple.

Many research findings in large animal reproductionduring the past 30 years can be credited entirely to gray-scale UI. The literature has become so voluminous onhorses and cattle that some of the sections in this reviewhave been reduced to a selected list of ultrasound-basedresearch contributions that illustrate the research powerof ultrasound technologies in reproductive biology; thelists are not comprehensive. In addition, preference is givento the apparent original reports for historical purposes. Thereview heralds examples of the capability of UI for datacollection and statistical testing of hypotheses by re-searchers and as a source of enlightenment and fascinationfor all who watch the moving images. It is also entertainingto see something for the first time, even when it is anillusion (Fig. 1). Equine embryo mobility is featured as anexample of a discovery that was solely a product of trans-rectal gray-scale UI. The discovery can be consideredfundamental in that it extended into unexpected areas.Consideration will also be given to research findings bygray-scale UI in females of large species other than horsesand cattle, specialized research techniques, handling ofresearch data, and the application of UI to research in male

Fig. 1. Examples of image illusions that serve to enliven discussion amongobservers. (A) Cross sections of the rear hooves of a 200-day equine fetusand the surrounding tissues of the uterine horn. (B) Segment of the um-bilical cord of a 70-day equine fetus. The echoic area below the “poodle” is aportion of the fetus. (C) The agonizing ghostly face is an equine CL. (D) Thearrows delineate a cross section of the pelvic urethra of a stallion duringejaculation. Dilations of the vesicular-gland ducts (eyes) and urethral lumen(mouth) occur rhythmically with each ejaculatory pulse. Adapted from [2,3]with permission.

genitalia. Color-Doppler UI and Doppler ultrasonographyare also discussed. The term ultrasonography refers exclu-sively to the production of Doppler graphs that representthe changing blood velocities within an arterial pulse andtherefore will be distinct from color Doppler UI.

Applied and diagnostic uses of UI in reproductivemanagement in horses, cattle, and other large domesticspecies [3–12] and the principles, equipment, and tech-niques [2,4,13] have been well-reported and are notconsidered. The review updates and expands on previousreviews on research applications of gray-scale UI in horsesand cattle [2,3,6,14,15]. This review is part of the 40-yearcommemorative issue of Theriogenology and notes therole of the journal in disseminating the initial (Table 1) andmounting reports on UI and ultrasonography.

2. Gray-scale UI in mares

In the early 1980s, there was a rapid increase in trans-rectal gray-scale UI by equine theriogenologists [16–19].Horse-breeding farms were a natural site for initiation ofthe technology for several reasons: (1) intensive veterinarymanagement of reproduction in individual mares, (2)justification for purchase of relatively expensive equip-ment, and (3) fluid-filled embryonal and fetal conceptusand the noncoiled shape of the uterus permitted intriguingdisplays of the dynamic interactions between the conceptusand uterus. The transrectal route for insertion of the ultra-sound transducer in the early years came naturally becauseof the well-established transrectal route for routine tactileexamination of the internal reproductive organs in mares.

The linear intrarectal transducers that were used formares in 1980 to 1982 were of low frequency (e.g., 3.0MHz). The low resolution of the original transducers isapparent in published Polaroid ultrasonograms of equineembryos from Days 11 to 48 [20]. In 1983, a 5.0-MHz lineartransducer was introduced [13]. The 5.0-MHz transducerhad less depth of penetration, but the greater resolutionyielded detailed information on structures close to the

Table 1Apparent milestones in the introduction of ultrasound technologies forresearch study of the reproductive organs in large animals.a

Year Ultrasound technologyb

1980 UI of ovaries and uterus in mares [1]1983 5.0 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]1994–7 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 2004.Abbreviations: UI, ultrasonic imaging.

a Some of these research milestones were preceded by reports on theclinical use of ultrasound for pregnancy diagnoses in sheep, goats, andpigs (reviewed in [12]).

b References in italics indicate publication in Theriogenology.

O.J. Ginther / Theriogenology 81 (2014) 112–125114

transducer and separated only by the wall of the rectum.Consequently, published ultrasonograms of the echo-texture of reproductive structures of mares were superiorto those that were available for women, who at the timewere being scanned through the abdominal wall [2]. Sub-sequently, this disadvantage was negated with the adap-tation of transvaginal transducers for women. Theultrasound scanners used in the early 1980s had analogscan converters; therefore, filming the scans was not asimple option [2]. By 1986, digital scan converters becameavailable and recording the scans and assessing the scansby computerized image analyses increased the researchpower of UI.

Equine reproductive physiologists were alerted to thetremendous potential of gray-scale UI for their own studiesby the original publication in 1980 [1] but were frustratedby equipment cost. Our laboratory was fortunate to begiven the loan of a high-quality ultrasound scanner with ananalog converter in 1982. The scanner screen became awindow to knowledge for our laboratory. Approximately 35publications (63% in Theriogenology) from 1983 to 1985based entirely on gray-scale UI contributed the followingexamples of original research findings: (1) UI dynamics ofthe follicles and CL throughout the estrous cycle [21]; (2) UIanatomy and pathology of the uterus [22]; (3) echotextureof the endometrial folds as an indicator of estrogen expo-sure for detecting the preovulatory period (Fig. 2) [22,23];(4) traveling of the spherical (6–16 mm) embryonic vesicleto all parts of the uterus (embryomobility) on Days 11 to 14or 15 (Day 0 ¼ ovulation) [24–26] and fixation on Day 15 or16 [19] in ponies and horses, respectively; (5) interactions

Fig. 2. Contrast between estrus and diestrus in the echotexture of uterinehorns in mares (cross sections) and heifers (longitudinal sections). The im-ages in heifers are from the curvature at the cranial portion of a horn; thearrows delineate the mesometrial attachment. Adapted from [3,6] withpermission.

between twins during embryo mobility [27]; (6) a tech-nique for manual elimination of one member of a twin setduring embryo mobility [28]; (7) physical nature of em-bryonic death [29]; and (8) postfixation mechanism forspontaneous embryo reduction of one member of a twinset (Fig. 3) [30]. The knowledge windfall culminated in thefirst text and reference book on gray-scale UI in animals in1986 under the narrow title of “Ultrasonic Imaging andReproductive Events in the Mare” [31]. It was the mostexciting 3 years of my ongoing 50 years of research.

Ultrasound-based contributions in mares after the 1986book included many studies on ovarian follicles. After theinitial characterization of follicle population dynamicsbased on diameter groupings [21], the technique of iden-tifying individual follicles from examination to examina-tion was introduced by researchers in New York [32].Monitoring follicles became essential for research on thehormonal mechanisms associated with follicular waves,selection of a dominant follicle, and hormonal preparationof the ovulatory follicle [33–35]. Follicle dynamics werecharacterized for the prepubertal stage [36], ovulatoryseason [35,37], anovulatory season [38], during old age[39], and in comparison of miniature ponies and largerbreeds [40]. It was also found that the characteristics of theovulatory follicular wave are surprisingly similar betweenmares and women, except for the 2.2-fold larger follicle inmares at specific events during a wave [41,42]. A majoradvance was the finding that both minor waves (waveswithout a dominant follicle) and major waves occur inmares [43], and the emergence of each wave type isattributable to a surge in FSH concentration [44].

Original ultrasound-based findings in mares that did notinvolve follicular waves after the 1986 book included: (1) firstnoninvasive characterization and quantification of uterinecontractions in any species [45]; (2) factors affecting contrac-tility and role of uterine contractions in embryomobility [26];(3)first noninvasive continuous characterization and timingof

Fig. 3. Process of embryo reduction in twin equine embryos with fixation inthe same uterine horn. On Day 17, the thick vascular wall (yellow line) of theyolk sac of the doomed vesicle (Y2) is forced by uterine massage into thethin nonvascular wall of the yolk sac of the thriving vesicle (Y1); the vasculararea (red line) of Y1 is shown in contact with the endometrium. Similarly, onDay 29 the vascular wall of the doomed vesicle (yellow line) is blocked fromthe 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.

Fig. 5. Examples of fetal mobility in a mare and heifer. The equine fetus isshown with head (HD) and chest (CH) on the allantoic floor and 4 s laterbeing propelled into the allantoic fluid by a vigorous neck (NK) arch. Theamniotic membrane is indicated by arrows. The bovine fetus has done a flip-flop from side to side as indicated by the rumen (RU) changing from theupper to lower position. Adapted from [3,6] with permission.

O.J. Ginther / Theriogenology 81 (2014) 112–125 115

follicle evacuation during ovulation in any species [46]; (4)development of fetal gender diagnosis on Days 50 to 60 byrelative 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 self-inflicted damage [48]; (6) changes in echotexture, especiallythat an anechoic layer of the follicle wall distinguishes thefuture dominant follicle before the diameter manifestation offollicle selection (deviation) [49]; (7) discovery of serration ofthe granulosa layer of the preovulatory follicle and its use forpredicting imminent ovulation [50]; (8) incidence and natureof embryonic death, including expulsion through the cervix[29,51]; (9) characterization of the natural outcome of fetaltwins and in vivo identification of fetal twins in late gestationby 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) onfertility [53] and the conclusion that a small fluid collection(diameter, 5–20 mm) during diestrus and detectable only byUI is an indicator ofmetritis [29,53]; (11) incidence and natureof dysorientation of the embryo proper and spontaneouscorrection [54]; (12) accumulation of fluid in the infundib-ulum during the estrous cycle [55]; (13) effects of age onuterine function [56]; (14) assessment of fetal fluids, placentalthickness [57], and ascending placentitis [58]; and (15) dis-covery that fetal (after Day 40) activity (Fig. 5) involvedfrequent changes in orientation and in location among theuterine horns and body, allantoic fluid shifts throughout theuterus, closing and opening of parts or all of each uterine horn,and closure of both horns at mid-pregnancy with both fetalhind limbs encased by the umbilical cord horn [59–61].Excluding the many research reports involving follicle dy-namics, 24 findings or characterizations in mare reproductivephysiology are listed that likely would still be unknown orunclarified if it were not for the noninvasive but powerfulultrasound technology of transrectal gray-scale UI. Thesefindings and techniques give credence to the opinion enun-ciated in the title of this review. In addition to monitoringfollicles and detecting ovulation, nonfollicular research

Fig. 4. Frontal views across the front limbs (FL), hind limbs (HL), umbilicalcord (UC), and tail (T) of a bovine fetus at approximately Day 60. The po-sition of the genital tubercle (GT) is between the hind limbs in the male fetusand caudal to the hind limbs in the female fetus. Adapted from [6] withpermission.

findings by UI account for the availability of techniques thatcan be used by equine clinicians. These are indicated above bypoints 3 and 6 for 1983 through 1985 and points 4, 7, 9, and 10during and after 1986.

3. Equine embryo mobility

Some of the discoveries in mares that are attributable totransrectal 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 anequivalent of 10 to 20 times per day on Days 11 to 15 [25,26]was a stark departure from the assumption that the embryoat this time was nestled snugly among the endometrialfolds. As the encapsulated embryo moves, it carries mes-sages for interaction with the uterine wall (Fig. 6). Onemessage stimulates myometrial contractions, which pro-vide the propulsive force for the embryo [26,62], andanother stimulates an increase in vascular perfusion in theuterine wall that moves approximately every 0.5 hour fromhorn to horn with the embryo [63]. The essentiality ofembryo mobility involves a message that is carried to allparts of the uterus to block the uterine luteolytic mecha-nism, ensuring the required maintenance of theprogesterone-producing CL [64]. All of the uterus must bevisited by the relatively small embryo because in this spe-cies the luteolytic signal from uterus to CL is through sys-temic channels [65]. On the days of mobility, a substance isdistributed that gradually stimulates increased uterine tone[62,66]. By the time the blockage of luteolysis is complete

Fig. 6. Sequence (1–4) of events associated with equine embryo mobility,uterine contractions, blockage of luteolysis during the first luteal response topregnancy, decrease in uterine diameter from an increase in tone, and fixationat a flexure in a caudal uterine horn. Adapted from [162] with permission.

O.J. Ginther / Theriogenology 81 (2014) 112–125116

(mean, Day 16), the embryonic vesicle has grown anduterine tone has increased (decreased uterine diameter) tothe point that fixation occurs at the flexure in a caudal hornthat offers the greatest impediment to continued mobility[26,67].

Embryo mobility also provided explanation or rationalefor hypotheses on several phenomena that were perplexingto equine veterinarians and biologists [68], including: (1)lack of agreement between side of ovulation and side offixation (embryo mobility involves both horns with noaffect of side of CL), (2) preferential fixation in postpartummares in the formerly nongravid horn (nongravid horn issmaller), (3) fixation almost always in the caudal portion ofone of the uterine horns (bend or impediment to mobilityin caudal horn), (4) fixation 1 day earlier in ponies than inhorses (uterus is smaller in ponies but embryonic vesicle isnot), and (5) greater incidence of unilateral than bilateralfixation of twins (first embryo to become fixed is animpediment to mobility by the other). Ultrasound scannerswere the window to the phenomenon of embryo mobilityand the means for characterizing the underlying mecha-nisms. Furthermore, embryo mobility and fetal activity andinteractions with the uterus are spectacular events thatinspire awe and respect for animal biology. With regard tothe impact on laymen as well as professionals, a survey byEQUUS magazine credited embryo mobility as anoutstanding discovery in equine research [69].

4. Gray-scale UI in heifers/cows

The first reports on transrectal gray-scale UI for exam-ining the reproductive tract of heifers/cows appeared in a

report from France on pregnancy diagnosis in 1982 [70] andreports on the echotexture of the reproductive organs andconceptus appeared in Theriogenology in 1984 [71–73].Apparently, the greatest current use of UI in cattle in mostlaboratories is to determine the time of ovulation as areference point or end point and to diagnose pregnancy.However, the technology has many more uses and poten-tial. Detailed studies have been done on follicle dynamicsbefore puberty [74], during the estrous cycle [75], duringearly [76] and late pregnancy [77], and during super-stimulation [73]. The technology has become essential forcollection of follicle data in cattle, as for mares, especiallyby maintaining the identity of follicles from examination toexamination [78]. The follicles have been studied during awide array of research projects involving many aspects offollicle dynamics, waves, and selection [33,79]. Studies withfollicles have included the demonstrations that the emer-gence of each follicular wave during the estrous cycle [80]and throughout pregnancy [77] is associated with a surgein FSH and that a close two-way coupling is involved in thefollicle/FSH relationship [81,82]. A recent study usedtransrectal UI of follicles to study roles and controls of thepreovulatory and periovulatory FSH surges [83]. Echo-texture reflects the function and endocrine status for thefollicular antrum and wall and for the CL [84–86]. Charac-terization of the nature and timing for evacuation ofapproximately 90% of the antral fluid during ovulationinvolved 4 s [87], comparedwith 60 s in mares [46]. Studieson follicle dynamics and selection have included otherbovine species such as Bos indicus [88] as well as B taurus;for example, deviation in the first postovulatory wave inNelore heifers and cows occurs approximately 1 day earlierwhen the future dominant follicle is 2 mm smaller than inHolsteins.

Research findings in cattle based on transrectal gray-scaleUI have included nonfollicular structures and events as fol-lows: (1) changes in size and echotexture of the CL [73],leading to comparisons of functional and structural CLchanges [89] and to the use of UI to define a change in CL area(cm2) as a reference point for initiating experiments [90]; (2)ultrasonic appearance of the uterus, including a more het-erogeneous echotexture during estrus (Fig. 2) [91]; (3)reduced curling of the uterine horns during estrus [91], un-like the apparent curling during estrus when based ontransrectal palpation; (4) characterization of uterine con-tractions during the estrous cycle and pregnancy [92]; (5)pregnancy characterization [72,93], including detailed de-scriptions of the conceptus on Days 10 to 60 [94–96]; (6)nature of embryonal and fetal loss [97–99] and factorsinvolved in the loss [100]; (7) determining fetal sex by rela-tive location of the genital tubercle (Fig. 4) [101] and later byidentification of genitalia [102]; (8) transient filling andemptying of various segments of the uterus by the fluid-filledplacental membranes, gradual increase in intrinsic activity ofthe fetus betweenDays 40 to 60, and changes in recumbency,presentation (cranial vs. caudal direction), and intrauterinelocations throughout pregnancy (Fig. 5) [6]; (9) assessinguterine inflammation [103] and postpartum ovarian activityand uterine involution [104,105]; (10) detection of twins [99]and spontaneous loss [106], and ultrasound-guided reduc-tion of one member of a twin set [107,108]; and (11)

O.J. Ginther / Theriogenology 81 (2014) 112–125 117

discovery that several fetal anomalies can be detected by UIon Days 55 to 120, including compact fetal mass, schistoso-mus reflexus, and fetal hydrops [6]. Early detection (e.g.,during fetal gender determination) and termination of thepregnancy is an advantage of UI in that many of theseanomalies result in dystocia at term. Although bovine re-searchers and clinicians were slower than their equinecounterparts to embrace gray-scale UI, the eventual resultsfurther entrench the accolades in the title of this review.

5. Gray-scale UI in other large species

The experiences with horses and cattle encouraged theuse of transrectal UI for evaluating the reproductive organs insheep, goats, pigs, llamas, water buffalo, and nondomesticlarge animals. The transabdominal route may be used insmaller species (e.g., sheep), especially for pregnancy diag-nosis [2], but the resolution of transrectal UI is needed fordetailed studies. For transrectal UI, a transducer extension ora personwith a small armmay be needed for rectal insertionand manipulation of the transducer [2]. An extension mayalso 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], studyingthe association between emergence of follicular waves andFSH surges [111], and determining follicle dynamics withhigh and low ovulation rates [112]. Pregnancy and follicularwaves 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 hornsduring estrus and diestrus, respectively [117]; (2) follicularwaves, including growth and regression of individual folli-cles [118]; and (3) echotexture of the CL [119]. Subsequentstudies in Canada in llamas have utilized UI as an aid intransvaginal collection of oocytes [120] and for a series ofstudies that culminated in the first demonstration of anovulation-inducing factor in the seminal plasma of llamas,alpacas, and bulls [121]. Follicular waves have also beenshown by UI to occur in water buffalo [122,123] and haveimplications for assisted reproduction [124].

Although slow to be adopted, transrectal scanners arewithout parallel in the study of the reproductive organs ofnondomesticated large animals. The knowledge gained onthe reproductive system through transrectal UI and otherultrasound techniques (e.g., oocyte collection) have shownpotential in aiding these species to propagate, especiallythose that are endangered. In farmed red deer, transrectalUI was used in 1990 to establish criteria for predictingcalving data by measurements of the uterus, amniotic sac,placentomes, and several parts of the fetus [125]. Theintroductory publication on transrectal UI for large zooanimals appeared in 1991 and provided descriptions andultrasonograms of the internal reproductive tract in Asianand African elephants, black rhinoceros, white rhinoceros,banteng, gaur, giraffe, and bactrian camel [126]. In ele-phants, ultrasonically detected changes in the cervix anduterus were described. Subsequent studies in Germanyusing transrectal UI have elucidated many of the unusualaspects of the reproductive cycle of the elephant that wereunknown before UI became available [127]. The elephant

was shown to be monovulatory with two follicular wavesduring the follicular phase. Recent studies have includedcolor Doppler and three-dimensional UI to describe lutealand conceptus development in elephants [128]. In theoriginal study [126], a nontranquilized rhinoceros was usedto identify individual ovarian follicles for 34 days and anultrasonogram of a 27-day embryo was recorded. The em-bryo of the rhinoceros resembled an equine embryo at asimilar stage and was in a similar location at the caudaluterine horn. Ultrasound-guided oocyte recovery in therhinoceros has also been reported [129]. The status of gray-scale UI in the rhinoceros and elephant and the role of UI inassisted reproduction have been reviewed [130].

Researchers in Canada have used transrectal UI to studythe ovaries in camels [131] and in several large species ofwildlife; approximately 50% of the reports were in Ther-iogenology. The ovaries of elk (wapiti) were studied in detailfor the anovulatory and ovulatory seasons [132–134],leading to programs for ovarian synchronization, inductionof follicular waves, and superovulation. Superstimulation offollicles, oocyte collection, embryo transfer, and ovariansynchronization programs have also been developed forbison 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 gray-scale UI.

6. Specialized gray-scale UI techniques

Ultrasound technologies have become increasinglyimportant not only for viewing and characterizing theimages of moving events (e.g., uterine contractions) andslow events (e.g., follicle growth), but also as a crucialcomponent or guide for specialized techniques. The listingof the following techniques is intended as an overview,and preference is given for the reports that apparentlyintroduced or popularized a technique: (1) first use ofultrasonically simulated biological structures (equineembryos) for reproduction research in any species [26];(2) computerized pixel analyses of ovarian structures inmares [139,140], heifers [84–86,141,142], and ewes [143];(3) observing images of semen streaming into the uterusas an aid for teaching and evaluating AI technicians [144];(4) first attaching of echogenic markers in reproductionresearch in any species (horses [145]); (5) guiding can-nulation into the caudal vena cava for sampling blood witha greater proportion of ovarian effluent in heifers [146];(6) inserting a research substance directly into a follicle inmares [147] and heifers [148]; (7) sampling follicular fluidin heifers [149] and mares [150]; (8) sequential biopsy ofthe CL in heifers [151] and mares [152]; (9) aspiration offollicle contents for functional follicle ablation in mares[153] and heifers [154]; (10) recovering and transferringoocytes into other follicles by transvaginal UI in cattle[155] and mares [156] aided by manual transrectalmanipulation of an ovary; (11) studying the develop-mental patterns of small follicles (1–3 mm) by transrectalUI in cattle [157]; (12) ultrasonic biomicroscopy for in vivoimaging of surface follicles as small as 0.4 mm (includingthe cumulus–oocyte complex) in cattle, using an ultra-sonic biomicroscope with a single crystal that emits

O.J. Ginther / Theriogenology 81 (2014) 112–125118

ultrasound waves of 20 to 70 MHz [158]; (13) evaluatingmoving structures such as the fetal heart by M-mode(motion mode) [2]; (14) transvaginal ultrasound guidingfor inseminating and transferring embryos directly into auterine horn by bypassing the cervix [159]; and (15)aspirating oocytes by transvaginal ultrasound scanningand guiding for in vitro fertilization [160]. Thus, the use ofspecialized techniques further enhances the prestige of UIas a boon to research. On the applied side, the in vivo re-covery of oocytes has become common in the embryotransfer industry subsequent to its publication in Ther-iogenology by researchers in The Netherlands [160,161].

7. Color-Doppler UI and ultrasonography

Doppler ultrasound is not used as frequently as gray-scale ultrasound and therefore the principles of theDoppler technology are discussed briefly. The Dopplertechnology is based on Doppler-shift frequencies, whereinthe frequency of echoes from moving red cells is increasedor decreased as the cells move toward or away from thetransducer [4,162,163]. The Doppler effect of ultrasound issimilar to the Doppler effect of sound, wherein the soundfrequency changes as the source (e.g., car horn) moves to-ward or away from a listener. Color-Doppler UI and Dopplerultrasonography provide distinctively different approachesfor assessing the vascular system of reproductive organs. Incolor-flow mode (Doppler UI), the direction of blood flowrelative to the face of the transducer is represented bydifferent colors on the screen display (Fig. 7). The extent ofcolor can be estimated by percentage of a tissue with colorsignals or can be calculated by computer, based on numberof colored pixels.

In spectral mode (Doppler ultrasonography), blood flowfor a focused location in a specific artery is assessed byplacing a sample-gate cursor (e.g., 1 mmwide) on the gray-

Fig. 7. Ultrasonograms from gray-scale UI and color Doppler UI of a longi-tudinal section of the spermatic cord of a bull. The gray-scale image showsanechoic sections of the highly convoluted testicular artery as it intertwineswith the network of veins of the pampiniform plexus. Some of the arterialsections are rounded (arrows) with specular reflections on the upper andlower surfaces, indicating a smooth arterial wall and a perpendicular di-rection of the ultrasound beams. The color-Doppler image shows arterialblood in red or blue depending on the direction of blood flow relative to theultrasound beams. Arterial cross sections that are devoid of color (arrows)indicate that the ultrasound beams intersected the blood flow at a 90� angleso that the direction of flow was neither away nor toward the transducer.Adapted from [162] with permission.

scale or color-mode image of the lumen of an artery (Fig. 8).The focused results from the sample gate are displayed onthe screen by a graph that represents changing blood flowvelocities at various times within a cardiac cycle or indi-vidual arterial pulse. The numerical velocities arecomputed and displayed on the screen for a selected car-diac cycle. Doppler indices (resistance index; pulsatilityindex) are also displayed on the screen. The indices areratios computed from the various points of the changingvelocities in the cardiac cycle. They are especially useful forthe tortuous arteries of the reproductive tract because theyare independent of the angle of the transducer to the angleof blood flow. These indices reflect the hemodynamics ofthe tissue supplied by the artery distal (downstream) to thesample gate. An increase in the resistance index or thepulsatility index indicates an increase in resistance andtherefore a decrease in vascular perfusion of the tissues.Techniques for locating an image of a cross-section of theuterine and ovarian arteries in mares [162,164] and heifers/cows [162,164,165] have been described. The locations ofthe pelvic arteries to the genitalia of stallions and bulls arealso illustrated [162].

The use of transrectal Doppler ultrasonography forresearch studies in large animal reproduction was first re-ported by researchers in Germany in a series of publicationsin Theriogenology, beginning in 1998 in mares [164] and in2000 in cows [165]. Ovarian and uterine blood flow ve-locities during the estrous cycle and uterine and umbilicalblood flow during pregnancy were included. Their studieson blood velocity spectral graphs in cattle during theestrous cycle, pregnancy, and postpartum have beenreviewed [163]. Vascular perfusion of the CL and uterus wasgreater during the first versus second follicular wave of theestrous cycle [166]. Color-Doppler displays of blood-flowsignals in the follicle and CL in cattle have been popularizedby researchers in Japan [167–170].

The following research findings and conclusions areprimarily from our laboratory and are used to illustrate thepower of color Doppler UI and ultrasonography in repro-duction research. Findings in mares include: (1) duringfollicle selection, blood flow in the follicle wall begins toincrease in the future dominant follicle compared with thelargest future subordinate follicle an equivalent of 1 daybefore diameter deviation [171] and is similar to the day ofincreasing prominence of the anechoic band [49]; (2) per-centage of follicle circumference with blood flow signalsbegins to decrease in the follicle wall 4 hours beforeovulation [172]; (3) serration of the granulosa opposite tothe future site of ovulation results from blood vesselsbeneath the granulosa [173]; (4) blood flow in the wall ofthe dominant follicle (>30 mm) is less in anovulatory fol-licles than in ovulatory follicles during the transition be-tween anovulatory and ovulatory seasons [174]; (5)percentage of wall of the preovulatory follicle with bloodflow signals is greater in mares that subsequently becomepregnant than for those that do not [175]; (6) percentage ofthe circumference of a preovulatory follicle with blood flowsignals is greater and includes the apical area (site of po-tential rupture) when a hemorrhagic anovulatory follicleforms than when the follicle is ovulatory [176]; (7) neitheran increase nor decrease in luteal blood flow was detected

Fig. 8. Doppler ultrasonogram of changes in blood flow velocity during an individual arterial or cardiac pulse. The sample gate (SG) delineates the small focusthat 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 theplane of the ultrasound beams. Blood velocities of the systolic and diastolic portions of the cardiac cycle are depicted. Blood velocity is determined and listed onthe scanner screen at various points of the velocity graph or spectrum. In this example, the average velocity is shown and is called time-averaged maximumvelocity (TAMV). The resistance index is also shown. A greater index indicates less blood flow distal to the sample gate. Adapted from [162] with permission.

O.J. Ginther / Theriogenology 81 (2014) 112–125 119

before the beginning of a progesterone decrease in spon-taneous luteolysis [177]; (8) after follicle evacuation,vascularization of the CL begins at the basal area (site ofgranulosa serration in the preovulatory follicle) and pro-gressively extends toward the apical area during Days 0 to 6[173]; (9) color-Doppler signals apparently in the endo-metrium mark the future post-orientation site of theequine embryo proper well before the embryo proper isdetectable by gray-scale imaging [178]; (10) heart rate of anequine embryo on Days 24 and 27 was lower by more than3 standard deviations in an embryo that was lost on Day 31than in controls [162]; (11) reduced uterine vascularperfusion is associated with uterine cysts [179]; and (12)the movement of the conceptus back and forth betweenuterine horns during embryo mobility is accompanied by aback-and-forth transient increase in vascular perfusion ofthe endometrium [63], and at the end of mobility perfusionis greater in the horn of fixation [63,180].

Color-Doppler UI research findings in heifers/cowsinclude (1) the percentage of follicle wall with blood flowsignals increases synchronously with the initiation of the LHsurge [168]; (2) the percentage of wall of the preovulatoryfollicle with blood flow signals is greater in heifers thatsubsequently become pregnant [181]; (3) during luteolysis,blood flow in the CL increases during the ascending portionof each pulse of PGF2a metabolite, remains elevated for 2hours and then decreases [182]; (4) the increased CL bloodflow during PGF2a treatment is from the direct stimulationof PGF2a on the CL vasculature in heifers [183]; and (5)endometrial scores for an increase in the extent of vascularperfusion follow expansion of the allantochorion thr-oughout each uterine horn in cattle [184]. These examples ofresearch findings in mares and heifers highlight the capa-bilities of UI in reproduction research and would not havebeen possible without the availability of transrectal color-Doppler UI. A text and reference book with colored imagesand more detailed discussion has been published [162].

A study on vascular perfusion of the endometrium inpregnantmares is used to illustrate the approaches that canbe used in uterine blood flow studies by the Doppler

ultrasound technologies [63]. Vascular perfusion wasassessed by the estimated score for number of color signalsin a section of the endometrium, by computer-generatednumber of color pixels (color-Doppler UI), and by bloodvelocity in various parts of an arterial pulse in an artery inthe mesometrial attachment (Doppler ultrasonography).Each of the three approaches indicated similarity betweenthe nonpregnant and pregnant mares until Day 12. On Day13 of pregnancy, vascular perfusion by estimation of thepercentage of color signals or by computerized pixel countwas greater in the uterine horn with the mobile embryo.After fixation (end of embryo mobility), each of the twoassessments by color-Doppler UI and a lower pulsatilityindex in the assessment by ultrasonography indicatedgreater vascular perfusion in the horn of fixation than inthe opposite horn.

The heart beat of the equine embryo is detectable onDays 17 to 20 or approximately 2 days earlier by color-Doppler UI than for detection of the embryo proper bygray-scale UI [162]. Color-flow signals extend beyond theheart and into vessels of the membranes by Days 27 or 30.In cattle, color-flow signals extend into the vessels by Day28 and are detectable in the umbilical cord, aorta, and ca-rotid artery. Color Doppler images of the embryo (beforeDay 40) and fetus (after Day 40) are shown for horses andcattle (Fig. 9). Extensive series of color-Doppler images forthe follicles, CL, uterus, embryo, and fetus in horses andcattle and examples of determination of blood velocitiesand resistance indices by ultrasonography have been pub-lished [162].

8. Handling of research data

Despite the more than 30-year history of ultrasonictechnologies as research tools, there seems to be a reluc-tance to apply statistical analyses to data obtained by gray-scale or color-Doppler UI, except for data from studies offollicles and follicular waves. Many other quantitative endpoints are available through the imaging technologies[2,3,6] as indicated by the examples in previous sections.

Fig. 10. Examples of a quantitative measurement (height from dorsal toventral surface) and a subjective score (0 [nil] to 4 [maximum]) for a bovineuterus. Each end point can be subjected to statistical analyses for differencesamong days. Adapted from [6] with permission.

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, ductusvenosus; 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.

O.J. Ginther / Theriogenology 81 (2014) 112–125120

End points that can be quantified and compared amonggroups include: (1) diameter or other dimensions of theembryonic vesicle, embryo proper, and fetus and othermeasurable structures involving the conceptus; (2) diam-eter, area, or volume of the CL; (3) embryo and fetal heartrate; (4) extent of embryo mobility measured by distanceand time in mares; (5) diameter or other measures of theuterus (Fig. 10); (6) height or other measures of fluidpockets in the uterus; and (7) percentage of a structure(e.g., CL) with color pixels, indicating the extent of vascularperfusion. With regard to follicles, diameter is a convenientmeasure that is readily understood. However, there may beinstances when surface area (as calculated from a cursortracing of the periphery) may be more meaningful becausefunction is most related to the periphery of the antrum [2].Irregular structures (e.g., equine CL) can be traced for area(cm2) determination. Many scanners have convenient ca-pabilities for obtaining such measurements and displayingthe numerical results on the screen. Thus, ultrasoundquantitative data can be used for statistical comparisonsamong time or groups similar, for example, to data forchanges in hormone concentrations.

Scoring of end points from minimal to maximal (e.g., 1–4) is a useful technique for image characteristics that arenot amenable to quantifying by conventional measuringtechniques (Fig. 10) [2]. Scoring can be useful, for example,for relative amounts that cannot be easily measured, suchas intrauterine fluid, endometrial edema, extent of uterinecontractions, changes in uterine shape, and percentageestimate of a defined area with color signals. The scoringapproach facilitates profiling and statistically analyzingchanges and analyzing temporal relationships or correla-tions among the ultrasonically monitored events as well asamong other events, such as changes in hormone

concentrations. The scoring system is subjective in that theoperator’s judgment is used. Bias can be minimized byfilming the images so that scoring can be done withoutknowledge of source, or scoring can be done by a second

O.J. Ginther / Theriogenology 81 (2014) 112–125 121

operator who is unaware of the source or even the hy-pothesis under test. We have developed considerableconfidence in the scoring approach in our laboratorybecause of consistent results among experiments and in-dependent operators and the ease of analyzing and inter-preting results in relationship to known biologicalmechanisms.

Information from gray-scale and color-Doppler UI can bedigitized and analyzed by computer. Digitization is a proce-dure in which each pixel of an image is assigned a numericalvalue for brightness in gray-scale UI, or the number of coloredpixels is counted in color-Doppler UI [2]. Conversion of imageinformation to numerical data allows the use of quantitativestatistics for temporal characterizations or testing of hypoth-eses. However, the sophisticated processing does not elimi-nate bias. The operator retains the responsibility of selectingand delineating the areas to be processed, and these decisionsare notoriously subject to bias. Computer pixel analyses,therefore, can be dangerous and lull the operator into a falsesense of confidence. It is best if the operator selecting theimages or delineating the area for study does not know theexperimental group or source of the image. Pixel analyseshave been used, for example, to characterize day-to-daychanges in the intensity or echogenicity of the CL [86,140],quantify changes in echotexture [85] and shape changes in thepreovulatory follicle as ovulation approaches [139], andevaluate the extent of vascular perfusion [4,162]. Detailedinformation on statistical handling of ultrasonic images andpixel analyses in cattle [6] and horses [3] is available.

The images displayed on the ultrasound screen repre-sent interactions between the technology and living tissues[2]. Artifactual and factual echoes are produced, andconsequently, embarrassing errors may be published.Proper instrument adjustment and proper interpretation ofthe echoes on the ultrasound screen are crucial. Interpre-tation requires knowledge of the relationships betweentissues and echoes and the ability to differentiate betweentrue and artifactual responses. There are two types of gray-scale reflections (specular and nonspecular) that are pre-sented as echoes [2]. Artifactual echoes can be mistaken forechoes that represent actual structures. In addition tospecular echoes, certain tissue formations also cause ul-trasonic waves to bend (refract), bounce back and forth orre-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 structuresor changes. Artifacts are especially common during UI ofthe reproductive tract because of the many pockets ofbowel 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 thecardiac cycle in Doppler ultrasonography aremost commonin transrectal examinations and result from movements oftissues, the animal, or transducer. Animal restraint oracclimation and filter settings can be used to lessen theclutter. In this regard, detomidine sedation in horses andxylazine sedation in heifers affects blood flow in majorarteries (e.g., internal iliac) but an effect on local vascularperfusion in the ovaries and endometrium has not beendetected [185].

9. The male

Stallions and bulls have not been neglected in the utili-zation of transrectal and transcutaneous gray-scale and colorDoppler UI [3,6,162]. Transrectal gray-scale UI apparentlywas first used in stallions in 1987 in a description of theaccessory sex glands [186]. Researchers in Idaho describedthe changes in the accessory sex glands before and aftersexual preparation (teasing of mares) [187]. The vesicularglands became rounded after sexual preparation from theentry of vesicular gel, as indicated by an increased anechoicarea. The Idaho researchers also developed a unique systemfor transrectal UI of the sex glands during ejaculation[188,189]. Apparently for the first time in any species, imagesof the dynamics of the sex glandswere observed in real time.Rhythmic pulses (emissions) of the prostate secretionsoccurred before the onset of themanually determined penileand urethral rhythmic contractions or sequential ejaculatorypulses. Discharge of the prostate continued during the firstfew ejaculatory pulses. Rhythmic ampullary and prostaticactivity began a few seconds before the start of ejaculation.Ampullary fluid passed through the ducts in distinct bolusesbefore ejaculation, and prostate emission continued for thefirst few ejaculatory contractions. In contrast, the emission ofvesicular glands with accompanying pulses in the ductsoccurred after ejaculatory or urethral contractions wereunderway (Fig. 1). These ultrasonically determined patternsof emission are consistent with the order of appearance ofthe various portions of the seminal fluid in stallion ejacu-lates. The results demonstrated the power of UI in physicallyactive organs.

The technique for transcutaneous imaging of theexternal genitalia in stallions has been described [190]. Astandoff pad is used on the ventral surface of the testes forimaging the branches of the testicular artery. The centralvein can be imaged running longitudinally. Abnormalities(e.g., tumors, focal inflammation) can be imaged. Abnor-malities of the epididymis and the cause of scrotalenlargement (e.g., gravitated fluid, inguinal hernia, bloodclot) also may be detected by transcutaneous UI.

The genitals of bulls have also been examined by gray-scale UI. The examinations are effective for assessment ofstructure and for generating research data [6,191]. Reportson the initial imaging of the bull testis in vitro in 1987 [192]and in vivo in 1988 [193] and the ultrasonic appearance ofthe accessory sex glands [191] were first published inTheriogenology. Mean pixel intensities from ultrasonogramsof testes parenchyma indicate that gray-scale UI may be auseful noninvasive method for determining reproductivedevelopment in maturing bulls [194]. The accessory sexglands are imaged transrectally and the testes, epididy-mides, and the testicular cones, transcutaneously. Thevascular cone consists of the highly coiled testicular artery(Fig. 7) intertwining with the venous network the pampi-niform plexus. Recent reports concluded with the opinionthat the primary use of UI in the assessment of reproductivefunction in bulls is the detection of lesions in the testes andscrotum [195], but assessing breeding soundness is limited[196]. Recent reviews are available for the many publica-tions on monitoring reproductive function by UI in bulls[196,197].

O.J. Ginther / Theriogenology 81 (2014) 112–125122

10. Conclusions

Ultrasound technologies for research in reproductioninvolve the production of images by gray-scale UI andcolor-Doppler UI and production of spectral Doppler graphs(ultrasonography) of changing blood velocities within anarterial pulse. Many research findings and discoveriesduring the past 30 years resulted from the availability ofthese technologies. Quantitative data and discrete datafrom ranking or scoring can be generated by UI as for otherresearch techniques and statistically analyzed for differ-ences among treatments, times, or groups. Examples ofdiscoveries through ultrasound technologies are the em-bryo mobility phenomenon in mares by gray-scale UIand the increase in blood flow during luteolysis in associ-ation with the ascending portion of each individualPGF2a metabolite pulse in heifers by color-Doppler UI.There can be little doubt that transrectal gray-scale UI,color-Doppler UI, and Doppler ultrasonography haveexpanded and indeed revolutionized research in large an-imal reproduction.

Acknowledgments

Production of this manuscript was supported by theEutheria Foundation, Cross Plains, Wisconsin. The authorthanks John Kastelic for critiquing a presubmitted copy ofthe manuscript and Maria Hoffman for word processingand figure preparation.

References

[1] Palmer E, Driancourt MA. Use of ultrasonic echography in equinegynecology. Theriogenology 1980;13:206–16.

[2] Ginther OJ. Ultrasonic imaging and animal reproduction: book 1,fundamentals. Cross Plains, WI: Equiservices Publishing; 1995.

[3] Ginther OJ. Ultrasonic imaging and animal reproduction: book 2,horses. Cross Plains, WI: Equiservices Publishing; 1995.

[4] Ginther OJ, Utt MD. Doppler ultrasound in equine reproduction:principles, techniques, and potential. J Equine Vet Sci 2004;24:516–26.

[5] McKinnon AO, Squires EL, Vaala WE, Varner DD. Equine repro-duction, vol 2. Ames, IA: Wiley-Blackwell; 2011. p. 2003–162.

[6] Ginther OJ. Ultrasonic imaging and animal reproduction: book 3,cattle. Cross Plains, WI: Equiservices Publishing; 1998.

[7] Russel AJF. The application of real-time ultrasonic scanning incommercial sheep, goats and cattle production enterprises. In:Taverne MAM, Willemse AH, editors. Diagnostic ultrasound andanimal reproduction. Boston: Kluwer; 1989. p. 73–87.

[8] Fricke PM. Scanning the future––ultrasonography as a reproduc-tive management tool for dairy cattle. J Dairy Sci 2002;85:1918–26.

[9] Ribadu AY, Nakao T. Bovine reproductive ultrasonography: a re-view. J Reprod Dev 1999;45:13–28.

[10] DesCoteaux L, Gnemmi G, Colloton J. Practical atlas of ruminantand camelid reproductive ultrasonography. Ames, IA: BlackwellPublishing; 2010.

[11] Kauffold J, Althouse GC. An update on the use of B-mode ultraso-nography in female pig reproduction. Theriogenology 2007;67:901–11.

[12] Kähn W, Volkmann D, Kenney RM. Veterinary reproductive ul-trasonography. Hannover: Schlütersche; 1994.

[13] Ginther OJ, Pierson RA. Ultrasonic evaluation of the reproductivetract of the mare: principles, equipment, and techniques. J EquineVet Sci 1983;3:195–201.

[14] Griffin PG, Ginther OJ. Research applications of ultrasonic imagingin reproductive biology. J Anim Sci 1992;70:953–72.

[15] Ginther OJ. Research applications of ultrasonic imaging in repro-ductive biology: update 1994. In: Thacker PA, editor. Livestock

production in the 21st century. Saskatoon, Saskatchewan, Canada:University of Saskatchewan; 1994. p. 133–45.

[16] Chevalier F, Palmer E. Ultrasonic echography in the mare. J ReprodFertil Suppl 1982;32:423–30.

[17] O’Grady JP, Yeager CH, Findleton L, Brown J, Esra G. In uterovisualization of the fetal horse by ultrasonic scanning. Equine Pract1981;3:45–9.

[18] Rantanen NW, Torbeck RL, DuMond SS. Early pregnancy diagnosisin the mare using transrectal ultrasound scanning techniques: apreliminary report. J Equine Vet Sci 1982;2:27–9.

[19] Simpson DJ, Greenwood RE, Ricketts SW, Rossdale PD,Sanderson M, Allen WR. Use of ultrasound echography for earlydiagnosis of single and twin pregnancy in the mare. J Reprod FertilSuppl 1982;32:431–9.

[20] Ginther OJ. Fixation and orientation of the early equine conceptus.Theriogenology 1983;19:613–23.

[21] Ginther OJ, Pierson RA. Ultrasonic anatomy of equine ovaries.Theriogenology 1984;21:471–83.

[22] Ginther OJ, Pierson RA. Ultrasonic anatomy and pathology of theequine uterus. Theriogenology 1984;21:505–15.

[23] Hayes KE, Pierson RA, Scraba ST, Ginther OJ. Effects of estrous cycleand season on ultrasonic uterine anatomy in mares. Theriogenol-ogy 1985;24:465–77.

[24] Ginther OJ. Mobility of the early equine conceptus. Theriogenology1983;19:603–11.

[25] Leith GS, Ginther OJ. Characterization of intrauterine mobility ofthe early equine conceptus. Theriogenology 1984;22:401–8.

[26] Ginther OJ. Dynamic physical interactions between equine embryoand uterus. Equine Vet J Suppl 1985;3:41–7.

[27] Ginther OJ. Mobility of twin embryonic vesicles in mares. Ther-iogenology 1984;22:83–95.

[28] Ginther OJ. The twinning problem: from breeding to day 16.Proceedings of Am Assoc Equine Pract, 1983:11–26.

[29] Ginther OJ, Garcia MC, Bergfelt DR, Leith GS, Scraba ST. Embryonicloss in mares: pregnancy rate, length of interovulatory intervals,and progesterone concentrations associated with loss during days11 to 15. Theriogenology 1985;24:409–17.

[30] Ginther OJ. Postfixation embryo reduction in unilateral and bilat-eral twins in mares. Theriogenology 1984;22:213–23.

[31] Ginther OJ. Ultrasonic imaging and reproductive events in themare. Cross Plains, WI: Equiservices Publishing; 1986.

[32] Sirois J, Ball BA, Fortune JE. Patterns of growth and regression ofovarian follicles during the oestrous cycle and after hemi-ovariectomy in mares. Equine Vet J Suppl 1989;8:43–8.

[33] Beg MA, Ginther OJ. Follicle selection in cattle and horses: role ofintrafollicular factors. Reproduction 2006;132:365–77.

[34] Ginther OJ, Gastal EL, Gastal MO, Beg MA. Dynamics of the equinepreovulatory follicle and periovulatory hormones: what’s new? JEquine Vet Sci 2008;28:454–60.

[35] Jacob JC, Gastal EL, Gastal MO, Carvalho GR, Beg MA, Ginther OJ.Temporal relationships and repeatability of follicle diameters andhormone concentrations within individuals in mares. ReprodDomest Anim 2009;44:92–9.

[36] Nogueira GP, Ginther OJ. Dynamics of follicle populations andgonadotropin concentrations in fillies during 2–10 months of age.Equine Vet J 2000;32:482–8.

[37] Ginther OJ, Beg MA, Gastal MO, Gastal EL. Follicle dynamics andselection in mares. Anim Reprod 2004;1:45–63.

[38] Donadeu FX, Ginther OJ. Follicular waves and circulatingconcentrations of gonadotrophins, inhibin andoestradiol during theanovulatory season in mares. Reproduction 2002;124:875–85.

[39] Ginther OJ, Gastal MO, Gastal EL, Jacob JC, Siddiqui MA, Beg MA.Effects of age on follicle and hormone dynamics during the oes-trous cycle in mares. Reprod Fertil Dev 2008;20:955–63.

[40] Gastal EL, Gastal MO, Beg MA, Neves AP, Petrucci BPL, Mattos RC,et al. Miniature ponies: similarities and differences from largerbreeds in follicles and hormones during the estrous cycle. J EquineVet Sci 2008;28:508–17.

[41] Ginther OJ, Gastal EL, Gastal MO, Bergfelt DR, Baerwald AR,Pierson RA. Comparative study of the dynamics of follicular wavesin mares and women. Biol Reprod 2004;71:1195–201.

[42] Ginther OJ. The mare: a 1000-pound guinea pig for study of theovulatory follicular wave in women. Theriogenology 2012;77:818–28.

[43] Ginther OJ. Major and minor waves during the equine estrouscycle. J Equine Vet Sci 1993;13:18–25.

[44] Ginther OJ, Bergfelt DR. Associations between FSH concentrationsand major and minor follicular waves in pregnant mares. Ther-iogenology 1992;38:807–21.

O.J. Ginther / Theriogenology 81 (2014) 112–125 123

[45] Cross DT, Ginther OJ. Uterine contractions in nonpregnant andearly pregnant mares and jennies as determined by ultrasonog-raphy. J Anim Sci 1988;66:250–4.

[46] Townson DH, Ginther OJ. Duration and pattern of follicular evac-uation during ovulation in the mare. Anim Reprod Sci 1987;15:131–8.

[47] Curran S, Ginther OJ. Ultrasonic diagnosis of equine fetal sex bylocation of the genital tubercle. J Equine Vet Sci 1989;9:77–83.

[48] Ginther OJ. The nature of embryo reduction in mares with twinconceptuses: deprivation hypothesis. Am J Vet Res 1989;50:45–53.

[49] Gastal EL, Donadeu FX, Gastal MO, Ginther OJ. Echotexturalchanges in the follicular wall during follicle deviation in mares.Theriogenology 1999;52:803–14.

[50] Gastal EL, Gastal MO, Ginther OJ. Serrated granulosa and otherdiscrete ultrasound indicators of impending ovulation in mares. JEquine Vet Sci 2006;26:67–73.

[51] Ginther OJ, Bergfelt DR, Leith GS, Scraba ST. Embryonic loss inmares: incidence and ultrasonic morphology. Theriogenology1985;24:73–86.

[52] Ginther OJ, Griffin PG. Natural outcome and ultrasonic identifica-tion of equine fetal twins. Theriogenology 1994;41:1193–9.

[53] Adams GP, Kastelic JP, Bergfelt DR, Ginther OJ. Effect of uterineinflammation and ultrasonically-detected uterine pathology onfertility in the mare. J Reprod Fertil Suppl 1987;35:445–54.

[54] Ginther OJ, Silva LA. Incidence and nature of disorientation of theembryo proper and spontaneous correction in mares. J Equine VetSci 2006;26:249–56.

[55] Gastal EL, Jacob JCF, Gastal MO, Ginther OJ. Accumulation of fluid inthe infundibulum during the estrous cycle in mares. J Equine VetSci 2007;27:251–9.

[56] Carnevale EM, Ginther OJ. Relationships of age to uterine functionand reproductive efficiency in mares. Theriogenology 1992;37:1101–5.

[57] Renaudin CD, Troedsson MHT, Gillis CL, King VL, Bodena A. Ul-trasonographic evaluation of the equine placenta by transrectaland transabdominal approach in the normal pregnant mare.Theriogenology 1997;47:559–73.

[58] Renaudin CD, Troedsson MHT, Schrenzel MD. Transrectal ultraso-nographic diagnosis of ascending placentitis in the mare: a reportof two cases. Equine Vet Ed 1999;11:69–74.

[59] Ginther OJ. Equine physical utero-fetal interactions: a challengeand a wonder for the practitioner. J Equine Vet Sci 1994;14:313–8.

[60] Ginther OJ, Griffin PG. Equine fetal kinetics: presentation andlocation. Theriogenology 1993;40:1–11.

[61] Ginther OJ. Equine fetal kinetics: allantoic-fluid shifts and uterine-horn closures. Theriogenology 1993;40:241–56.

[62] Griffin PG, Carnevale EM, Ginther OJ. Effects of the embryo onuterine morphology and function in mares. Anim Reprod Sci 1993;31:311–29.

[63] Silva LA, Gastal EL, Beg MA, Ginther OJ. Changes in vascularperfusion of the endometrium in association with changes inlocation of the embryonic vesicle in mares. Biol Reprod 2005;72:755–61.

[64] McDowell KJ, Sharp DC, Grubaugh W, Thatcher WW, Wilcox CJ.Restricted conceptus mobility results in failure of pregnancymaintenance in mares. Biol Reprod 1988;38:335–42.

[65] Ginther OJ. Equine pregnancy: physical interactions between theuterus and conceptus. Proc AmAssoc Equine Pract 1998;44:73–104.

[66] Gastal MO, Gastal EL, Torres CA, Ginther OJ. Effect of PGE2 onuterine contractility and tone in mares. Theriogenology 1998;50:989–99.

[67] Gastal MO, Gastal EL, Kot K, Ginther OJ. Factors related to the timeof fixation of the conceptus in mares. Theriogenology 1996;46:1171–80.

[68] Ginther OJ. Reproductive biology of the mare: basic and appliedaspects. Second edition. Cross Plains, WI: Equiservices Publishing;1992.

[69] Kilby E. The evolution and revolutions of equine research. EQUUS1985;97:28–130.

[70] Chaffaux S, Valon F, Martinez J. Evolution de l’image échographi-que du produit de conception chez la vache. Bull Acad vét Fr 1982;55:213–21.

[71] Reeves JJ, Rantanen NW, Hauser M. Transrectal real-time ultra-sound scanning of the cow reproductive tract. Theriogenology1984;21:485–94.

[72] Pierson RA, Ginther OJ. Ultrasonography for detection of preg-nancy and study of embryonic development in heifers. Ther-iogenology 1984;22:225–33.

[73] Pierson RA, Ginther OJ. Ultrasonography of the bovine ovary.Theriogenology 1984;21:495–504.

[74] Evans ACO, Adams GP, Rawlings NC. Prepubertal ovarian andendocrine changes preceding first ovulation in heifers. J ReprodFert 1993;100:187–94.

[75] Pierson RA, Ginther OJ. Follicular population dynamics during theestrous cycle of the mare. Anim Reprod Sci 1987;14:219–31.

[76] Pierson RA, Ginther OJ. Ovarian follicular populations during earlypregnancy in heifers. Theriogenology 1986;26:649–58.

[77] Ginther OJ, Kot K, Kulick LJ, Martin S, Wiltbank MC. Relationshipsbetween FSH and ovarian follicular waves during the last sixmonths of pregnancy in cattle. J Reprod Fertil 1996;108:271–9.

[78] Sirois J, Fortune JE. Ovarian follicular dynamics during the estrouscycle in heifers monitored by real-time ultrasonography. BiolReprod 1988;39:308–17.

[79] Adams GP, Jaiswal R, Singh J, Malhi P. Progress in understandingovarian follicular dynamics in cattle. Theriogenology 2008;69:72–80.

[80] Adams GP, Matteri RL, Kastelic JP, Ko JCH, Ginther OJ. Associationbetween surges of follicle-stimulating hormone and the emer-gence of follicular waves in heifers. J Reprod Fert 1992;94:177–88.

[81] Ginther OJ, Bergfelt DR, Kulick LJ, Kot K. Selection of the dominantfollicle in cattle: role of two-way functional coupling betweenfollicle-stimulating hormone and the follicles. Biol Reprod 2000;62:920–7.

[82] Ginther OJ, Bashir ST, Rakesh HB, Hoffman MM. Two-way couplingbetween FSH and the dominant follicle in heifers. Theriogenology2013;80:463–9.

[83] Haughian JM, Ginther OJ, Diaz FJ, Wiltbank MC. Gonadotropin-releasing hormone, estradiol, and inhibin regulation offollicle-stimulating hormone and luteinizing hormone surges:implications for follicle emergence and selection in heifers. BiolReprod 2013;88:165.

[84] Singh J, Pierson RA, Adams GP. Ultrasound image attributes ofbovine ovarian follicles and endocrine and functional correlates. JReprod Fertil 1998;112:19–29.

[85] Tom JW, Pierson RA, Adams GP. Quantitative echotexture analysisof bovine ovarian follicles. Theriogenology 1998;50:339–46.

[86] Tom JW, Pierson RA, Adams GP. Quantitative echotexture analysisof bovine corpora lutea. Theriogenology 1998;49:1345–52.

[87] Kot K, Ginther OJ. Ultrasonic characterization of ovulatory follic-ular evacuation and luteal development in heifers. J Reprod Fertil1999;115:39–43.

[88] Sartorelli ES, Carvalho LM, Bergfelt DR, Ginther OJ, Barros CM.Morphological characterization of follicle deviation in Nelore (Bosindicus) heifers and cows. Theriogenology 2005;63:2382–94.

[89] Kastelic JP, Bergfelt DR, Ginther OJ. Relationship between ultra-sonic assessment of the corpus luteum and plasma progesteroneconcentration in heifers. Theriogenology 1990;33:1269–78.

[90] Pugliesi G, Khan FA, Hannan MA, Beg MA, Carvalho GR, Ginther OJ.Inhibition of prostaglandin biosynthesis during postluteolysis andeffects on CL regression, prolactin, and ovulation in heifers. Ther-iogenology 2012;78:443–54.

[91] Pierson RA, Ginther OJ. Ultrasonographic appearance of the bovineuterus during the estrous cycle. J Am Vet Med Assoc 1987;190:995–1001.

[92] Bonafos LD, Kot K, Ginther OJ. Physical characteristics of the uterusduring the bovine estrous cycle and early pregnancy. Ther-iogenology 1995;43:713–21.

[93] Chaffaux S, Reddy GNS, Valon F, Thibier M. Transrectal real-timeultrasound scanning for diagnosing pregnancy and for moni-toring embryonic mortality in dairy cattle. Anim Reprod Sci 1986;10:193–200.

[94] Curran S, Pierson RA, Ginther OJ. Embryonic loss and ultrasonicanatomy of the bovine conceptus on days 10 to 20. J Am Vet MedAssoc 1986;189:1289–94.

[95] Curran S, Pierson RA, Ginther OJ. Ultrasonic anatomy of the bovineconceptus ondays20 to60. J AmVetMedAssoc1986;189:1295–302.

[96] Kastelic JP, Curran S, Pierson RA, Ginther OJ. Ultrasonic evaluationof the bovine conceptus. Theriogenology 1988;29:39–54.

[97] Kastelic JP, Northey DL, Ginther OJ. Spontaneous embryonic deathon Days 20 to 40 in heifers. Theriogenology 1991;35:351–63.

[98] Kastelic JP, Ginther OJ. Fate of conceptus and corpus luteum afterinduced embryonic loss in heifers. J Am Vet Med Assoc 1989;194:922–8.

[99] Dobson H, Rowan TG, Kippax IS, Humblot P. Assessment of fetalnumber, and fetal and placental viability throughout pregnancy incattle. Theriogenology 1993;40:411–25.

O.J. Ginther / Theriogenology 81 (2014) 112–125124

[100] Lopez-Gatius F, Santolaria P, Yaniz J, Rutllant J, Lopez-Bejar M. Fac-tors affecting pregnancy loss fromgestationDay 38 to 90 in lactatingdairy cows from a single herd. Theriogenology 2002;57:1251–61.

[101] Curran S, Kastelic JP, Ginther OJ. Determining sex of the bovinefetus by ultrasonic assessment of the relative location of the gen-ital tubercle. Anim Reprod Sci 1989;19:217–27.

[102] Müller E, Wittkowski G. Visualization of male and female charac-teristics of bovine fetuses by real-time ultrasonics. Theriogenology1986;22:571–4.

[103] Kasimanickam R, Duffield TF, Foster RA, Gartley CJ, Leslie KE,Walton JS, et al. Endometrial cytology and ultrasonography for thedetection of subclinical endometritis in postpartum dairy cows.Theriogenology 2004;62:9–23.

[104] Kamimura S, Ohgi T, Takahashi M, Tsukamoto T. Postpartumresumption of ovarian activity and uterine involution monitored byultrasonography in Holstein cows. J Vet Med Sci 1993;55:643–7.

[105] Okano A, Tomizuka T. Ultrasonic observation of postpartum uter-ine involution in the cow. Theriogenology 1987;27:369–76.

[106] Lopez-Gatius F, Hunter RH. Spontaneous reduction of advancedtwin embryos: its occurrence and clinical relevance in dairy cattle.Theriogenology 2005;63:118–25.

[107] Andreu-Vazquez C, Garcia-Ispierto I, Lopez-Bejar M, de Sousa NM,Beckers JF, Lopez-Gatius F. Clinical implications of induced twinreduction in dairy cattle. Theriogenology 2011;76:512–21.

[108] Andreu-Vazquez C, Garcia-Ispierto I, Lopez-Gatius F. Manualrupture versus transvaginal ultrasound-guided aspiration ofallanto-amniotic fluid in multiple pregnancies: a clinical approachto embryo reduction in dairy cattle. J Reprod Dev 2012;58:420–4.

[109] Buckrell BC, Bonnett BN, Johnson WH. The use of a real-time ul-trasound rectally for early pregnancy diagnosis in sheep. Ther-iogenology 1986;25:665–73.

[110] White IR, Russel AJF, Fowler DG. Real-time scanning in the diag-nosis of pregnancy and the determination of foetal numbers insheep. Vet Rec 1984;115:140–3.

[111] Ginther OJ, Kot K, Wiltbank MC. Associations between emergenceof follicular waves and fluctuations in FSH concentrations duringthe estrous cycle in ewes. Theriogenology 1995;43:689–703.

[112] Gibbons JR, Kot K, Thomas DL, Wiltbank MC, Ginther OJ. Follicularand FSH dynamics in ewes with a history of high and low ovula-tion rates. Theriogenology 1999;52:1005–20.

[113] Lavoir MC, Taverne MAM. The diagnosis of pregnancy and pseu-dopregnancy, and the determination of foetal numbers of goats, bymeans of real-time ultrasound scanning. In: Taverne MAM,Willemse AH, editors. Diagnostic ultrasound and animal repro-duction. Boston: Kluwer; 1989. p. 89–96.

[114] Ginther OJ, Kot K. Follicular dynamics during the ovulatory seasonin goats. Theriogenology 1994;42:987–1001.

[115] Ryan DP, Yaakub H, Harrington D, Lynch PB. Follicular develop-ment during early pregnancy and the estrous cycle of the sow.Theriogenology 1994;42:623–32.

[116] Cartee RE, Powe Jr TA, Ayer RL. Ultrasonographic detection ofpregnancy in sows. Mod Vet Pract 1985;66:23–6.

[117] Adams GP, Griffin PG, Ginther OJ. In situ morphologic dynamics ofovaries, uterus, and cervix in llamas. Biol Reprod 1989;41:551–8.

[118] Adams GP, Sumar J, Ginther OJ. Effects of lactational and repro-ductive status on ovarian follicular waves in llamas (Lama glama). JReprod Fertil 1990;90:535–45.

[119] Adams GP, Sumar J, Ginther OJ. Form and function of the corpusluteum in llamas. Anim Reprod Sci 1991;24:127–38.

[120] Brogliatti GM, Palasz AT, Rodriguez-Martinez H, Mapletoft RJ,Adams GP. Transvaginal collection and ultrastructure of llama(Lama glama) oocytes. Theriogenology 2000;54:1269–79.

[121] Ratto MH, Huanca W, Singh J, Adams GP. Comparison of the effectof ovulation-inducing factor (OIF) in the seminal plasma of llamas,alpacas, and bulls. Theriogenology 2006;66:1102–6.

[122] Manik RS, Madan ML, Singla SK. Ovarian follicular dynamics inwater buffaloes (Bubalus bubalis): ultrasonically monitoring indi-vidual follicles for wave hypothesis. Theriogenology 1994;41:247.

[123] Baruselli PS, Mucciolo RG, Visintin JA, Viana WG, Arruda RP,Madureira EH, et al. Ovarian follicular dynamics during theestrous cycle in buffalo (Bubalus bubalis). Theriogenology 1997;47:1531–47.

[124] Campanile G, Baruselli PS, Neglia G, Vecchio D, Gasparrini B,Gimenes LU, et al. Ovarian function in the buffalo and implicationsfor embryo development and assisted reproduction. Anim ReprodSci 2010;121:1–11.

[125] Wilson PR, Bingham CM. Accuracy of pregnancy diagnosis andprediction of calving date in red deer using real-time ultrasoundscanning. Vet Rec 1990;126:133–5.

[126] Adams GP, Plotka ED, Asa CS, Ginther OJ. Feasibility of character-izing reproductive events in large nondomestic species by trans-rectal ultrasonic imaging. Zoo Biol 1991;10:247–59.

[127] Hildebrandt TB, Lueders I, Hermes R, Goeritz F, Saragusty J.Reproductive cycle of the elephant. Anim Reprod Sci 2011;124:176–83.

[128] Lueders I, Niemuller C, Rich P, Gray C, Hermes R, Goeritz F, et al.Gestating for 22 months: luteal development and pregnancymaintenance in elephants. Proc Biol Sci 2012;279:3687–96.

[129] Hermes R, Göritz F, Portas TJ, Bryant BR, Kelly JM, Maclellan LJ,et al. Ovarian superstimulation, transrectal ultrasound-guidedoocyte recovery, and IVF in rhinoceros. Theriogenology 2009;72:959–68.

[130] Hermes R, Göritz F, Streich W, Hildebrandt T. Assisted reproduc-tion in female rhinoceros and elephants–current status and futureperspective. Reprod Domest Anim 2007;42(Suppl. 2):33–44.

[131] Skidmore JA, Adams GP, Billah M. Synchronisation of ovarianfollicular waves in the dromedary camel (Camelus dromedarius).Anim Reprod Sci 2009;114:249–55.

[132] McCorkell RB, MacDougall L, Adams GP. Ovarian follicle develop-ment in wapiti (Cervus elaphus) during the anovulatory season.Theriogenology 2003;61:473–83.

[133] McCorkell RB, Woodbury MR, Adams GP. Ovarian follicular andluteal dynamics in wapiti during the estrous cycle. Theriogenology2006;65:540–56.

[134] McCorkell RB, Woodbury MR, Adams GP. Ovarian follicular andluteal dynamics in wapiti during seasonal transitions. Ther-iogenology 2007;67:1224–32.

[135] McCorkell RB, Woodbury MR, Adams GP. Serial ovarian ultraso-nography in wild-caught wood bison (Bos bison athabascae).Theriogenology 2013;80:552–6.

[136] Testa JW, Adams GP. Body condition and adjustments to repro-ductive effort in female moose (Alces alces). J Mammal 1998;79:1345–54.

[137] Hoare EK, Parker SE, Flood PF, Adams GP. Ultrasonic imaging ofreproductive events in muskoxen. Rangifer 1995;17:119–23.

[138] Adams GP, Testa JW, Goertz CEC, Ream RR. Ultrasonographiccharacterization of reproductive anatomy and early embryonicdetection in the northern fur seal (Callorhinus ursinus) in the field.Mar Mammal Sci 2007;23:445–52.

[139] Townson DH, Ginther OJ. Size and shape changes in the preovu-latory follicle in mares based on digital analysis of ultrasonic im-ages. Anim Reprod Sci 1989;21:63–71.

[140] Townson DH, Ginther OJ. Ultrasonic echogenicity of developingcorpora lutea in pony mares. Anim Reprod Sci 1989;20:143–53.

[141] Vassena R, Adams GP, Mapletoft RJ, Pierson RA, Singh J. Ultrasoundimage characteristics of ovarian follicles in relation to oocytecompetence and follicular status in cattle. Anim Reprod Sci 2003;76:25–41.

[142] Eramian MG, Adams GP, Pierson RA. Enhancing ultrasound texturedifferences for developing an in vivo ’virtual histology’ approach tobovine ovarian imaging. Reprod Fertil Dev 2007;19:910–24.

[143] Liu X, Hart EJ, Dai Q, Rawlings NC, Pierson RA, Bartlewski PM.Ultrasonographic image attributes of non-ovulatory follicles andfollicles with different luteal outcomes in gonadotropin-releasinghormone (GnRH)-treated anestrous ewes. Theriogenology 2007;67:957–69.

[144] Beal WE, Edwards RB, Kearnan JM. Use of B-mode, linear arrayultrasonography for evaluating the technique of bovine artificialinsemination. J Dairy Sci 1989;72:2198–202.

[145] Griffin PG, Ginther OJ. Dynamics of uterine diameter and endo-metrial morphology during the estrous cycle and early pregnancyin mares. Anim Reprod Sci 1991;25:133–42.

[146] Norman ST, Fields MJ. Ultrasound-guided cannulation of thecaudal vena cava in the bovine for selective sampling of ovarianeffluent. Theriogenology 1993;39:691–701.

[147] Gastal EL, Kot K, Ginther OJ. Ultrasound-guided intrafolliculartreatment in mares. Theriogenology 1995;44:1027–37.

[148] Kot K, Gibbons JR, Ginther OJ. A technique for intrafollicular in-jections in cattle: effects of hCG. Theriogenology 1995;44:41–50.

[149] Ginther OJ, Kot K, Kulick LJ, Wiltbank MC. Sampling follicular fluidwithout altering follicular status in cattle: oestradiol concentra-tions early in a follicular wave. J Reprod Fertil 1997;109:181–6.

[150] Gastal EL, Gastal MO, Wiltbank MC, Ginther OJ. Follicle deviationand intrafollicular and systemic estradiol concentrations in mares.Biol Reprod 1999;61:31–9.

[151] Kot K, Anderson LE, Tsai SJ, Wiltbank MC, Ginther OJ. Transvaginal,ultrasound-guided biopsy of the corpus luteum in cattle. Ther-iogenology 1999;52:987–93.

O.J. Ginther / Theriogenology 81 (2014) 112–125 125

[152] Beg MA, Gastal EL, Gastal MO, Ji S, Wiltbank MC, Ginther OJ.Changes in steady-state concentrations of messenger ribonucleicacids in luteal tissue during prostaglandin F2a induced luteolysisin mares. Anim Reprod Sci 2005;90:273–85.

[153] Gastal EL, Gastal MO, Bergfelt DR, Ginther OJ. Role of diameterdifferences among follicles in selection of a future dominant fol-licle in mares. Biol Reprod 1997;57:1320–7.

[154] Ginther OJ, Bergfelt DR, Kulick LJ, Kot K. Selection of the dominantfollicle in cattle: establishment of follicle deviation in less than 8hours through depression of FSH concentrations. Theriogenology1999;52:1079–93.

[155] Bergfelt DR, Brogliatti GM, Adams GP. Gamete recovery andfollicular transfer (graft) using transvaginal ultrasonography incattle. Theriogenology 1998;50:15–25.

[156] Carnevale EM, Ginther OJ. Use of a linear ultrasonic transducer forthe transvaginal aspiration and transfer of oocytes in the mare. JEquine Vet Sci 1993;13:331–5.

[157] Jaiswal RS, Singh J, Adams GP. Developmental pattern of smallantral follicles in the bovine ovary. Biol Reprod 2004;71:1244–51.

[158] Pfeifer LF, Siqueira LG, Adams GP, Pierson RA, Singh J. In vivo imagingof cumulus-oocyte-complexes and small ovarian follicles in cattleusing ultrasonic biomicroscopy. Anim Reprod Sci 2012;131:88–94.

[159] Silva LA, Gastal EL, Gastal MO, Jacob JCF, Reis CP, Ginther OJ. A newalternative for embryo transfer and artificial insemination inmares: ultrasound-guided intrauterine injection. J Equine Vet Sci2004;24:324–32.

[160] Pieterse MC, Kappen KA, Kruip TA, Taverne MA. Aspiration ofbovine oocytes during transvaginal ultrasound scanning of theovaries. Theriogenology 1988;30:751–62.

[161] Pieterse MC, Vos PL, Kruip TA, van Beneden TH, Willemse AH,Taverne MA. Transvaginal ultrasound guided follicular aspirationof bovine oocytes. Theriogenology 1991;35:19–24.

[162] Ginther OJ. Ultrasonic imaging and animal reproduction: book 4,color-Doppler ultrasonography. Cross Plains, WI: EquiservicesPublishing; 2007.

[163] Herzog K, Bollwein H. Application of Doppler ultrasonography incattle reproduction. Reprod Domest Anim Suppl 2007;42:51–8.

[164] Bollwein H, Maierl J, Mayer R, Stolla R. Transrectal color Dopplersonography of the A. uterina in cyclic mares. Theriogenology 1998;49:1483–8.

[165] Bollwein H, Meyer HH, Maierl J, Weber F, Baumgartner U, Stolla R.Transrectal Doppler sonography of uterine blood flow in cowsduring the estrous cycle. Theriogenology 2000;53:1541–52.

[166] Jordan A, Herzog K, Ulbrich SE, Beindorff N, Honnens A, Kruger L,et al. Genital blood flow and endometrial gene expression duringthe preovulatory period after prostaglandin F2a-induced luteolysisin different luteal phases in cows. J Reprod Dev 2009;55:309–15.

[167] Acosta TJ, Hayashi KG, Matsui M, Miyamoto A. Changes in follicularvascularity during the first follicular wave in lactating cows. JReprod Dev 2005;51:273–80.

[168] Acosta TJ, Hayashi KG, Ohtani M, Miyamoto A. Local changes inblood flow within the preovulatory follicle wall and early corpusluteum in cows. Reproduction 2003;125:759–67.

[169] Acosta TJ, Yoshizawa N, Ohtani M, Miyamoto A. Local changes inblood flow within the early and midcycle corpus luteum afterprostaglandin F2a injection in the cow. Biol Reprod 2002;66:651–8.

[170] Miyamoto A, Shirasuna K, Hayashi KG, Kamada D, Awashima C,Kaneko E, et al. A potential use of color ultrasound as a tool forreproductive management: new observations using color ultra-sound scanning that were not possible with imaging only in blackand white. J Reprod Dev 2006;52:153–60.

[171] Acosta TJ, Gastal EL, Gastal MO, Beg MA, Ginther OJ. Differentialblood flow changes between the future dominant and subordinatefollicles precede diameter changes during follicle selection inmares. Biol Reprod 2004;71:502–7.

[172] Gastal EL, Gastal MO, Ginther OJ. Relationships of changes inB-mode echotexture and colour-Doppler signals in the wall ofthe preovulatory follicle to changes in systemic oestradiol con-centrations and the effects of human chorionic gonadotrophin inmares. Reproduction 2006;131:699–709.

[173] Ginther OJ, Gastal EL, Gastal MO. Spatial relationships betweenserrated granulosa and vascularity of the preovulatory follicle anddeveloping corpus luteum. J Equine Vet Sci 2007;27:20–7.

[174] Acosta TJ, Beg MA, Ginther OJ. Aberrant blood flow area andplasma gonadotropin concentrations during the development ofdominant-sized transitional anovulatory follicles in mares. BiolReprod 2004;71:637–42.

[175] Silva LA, Gastal EL, Gastal MO, Beg MA, Ginther OJ. Relationshipbetween vascularity of the preovulatory follicle and establishmentof pregnancy in mares. Anim Reprod 2006;3:339–46.

[176] Ginther OJ, Gastal EL, Gastal MO, Beg MA. Incidence, endocri-nology, vascularity, and morphology of hemorrhagic anovulatoryfollicles in mares. J Equine Vet Sci 2007;27:130–9.

[177] Ginther OJ, Gastal EL, Gastal MO, Utt MD, Beg MA. Luteal bloodflow and progesterone production in mares. Anim Reprod Sci2007;99:213–20.

[178] Silva LA, Ginther OJ. An early endometrial vascular indicator ofcompleted orientation of the embryo and the role of dorsalendometrial encroachment in mares. Biol Reprod 2006;74:337–43.

[179] Ferreira JC, Gastal EL, Ginther OJ. Uterine blood flow and perfusionin mares with uterine cysts: effect of the size of the cystic area andage. Reproduction 2008;135:541–50.

[180] Bollwein H, Mayer R, Stolla R. Transrectal Doppler sonography ofuterine blood flow during early pregnancy in mares. Ther-iogenology 2003;60:597–605.

[181] Siddiqui MA, Almamun M, Ginther OJ. Blood flow in the wall of thepreovulatory follicle and its relationship to pregnancy establish-ment in heifers. Anim Reprod Sci 2009;113:287–92.

[182] Ginther OJ, Silva LA, Araujo RR, Beg MA. Temporal associationsamong pulses of 13,14-dihydro-15-keto-PGF2a, luteal blood flow,and luteolysis in cattle. Biol Reprod 2007;76:506–13.

[183] Ginther OJ, Beg MA. Dynamics of circulating progesterone con-centrations before and during luteolysis: a comparison betweencattle and horses. Biol Reprod 2012;86:1–12.

[184] Silva LA, Ginther OJ. Local effect of the conceptus on uterinevascular perfusion during early pregnancy in heifers. Reproduction2010;139:453–63.

[185] Araujo RR, Ginther OJ. Vascular perfusion of reproductive organs inpony mares and heifers during sedation with detomidine orxylazine. Am J Vet Res 2009;70:141–8.

[186] Little TV, Woods GL. Ultrasonography of accessory sex glands inthe stallion. J Reprod Fertil Suppl 1987;35:87–94.

[187] Weber JA, Geary RT, Woods GL. Changes in accessory sex glands ofstallions after sexual preparation and ejaculation. J Am Vet MedAssoc 1990;196:1084–9.

[188] Weber JA, Woods GL. A technique for transrectal ultrasonographyof stallions during ejaculation. Theriogenology 1991;36:831–7.

[189] Weber JA, Woods GL. Ultrasonographic measurement of stallionaccessory sex glands and excurrent ducts during seminal emissionand ejaculation. Biol Reprod 1993;49:267–73.

[190] Love CC. Ultrasonographic evaluation of the testis, epididymis, andspermatic cord of the stallion. Vet Clin North Am Equine Pract1992;8:167–82.

[191] Weber JA, Hilt CJ, Woods GL. Ultrasonographic appearance of bullaccessory glands. Theriogenology 1988;29:1347–55.

[192] Pechman RD, Eilts BE. B-mode ultrasonography of the bull testicle.Theriogenology 1987;27:431–41.

[193] Eilts BE, Pechman RD. B-mode ultrasound observations of bulltestes during breeding soundness examinations. Theriogenology1988;30:1169–75.

[194] Evans AC, Pierson RA, Garcia A, McDougall LM, Hrudka F,Rawlings NC. Changes in circulating hormone concentrations,testes histology and testes ultrasonography during sexual matu-ration in beef bulls. Theriogenology 1996;46:345–57.

[195] Kastelic JP, Brito LF. Ultrasonography for monitoring reproduc-tive function in the bull. Reprod Domest Anim Suppl 2012;47:45–51.

[196] Brito LF, Barth AD, Wilde RE, Kastelic JP. Testicular ultra-sonogram pixel intensity during sexual development and itsrelationship with semen quality, sperm production, and quan-titative testicular histology in beef bulls. Theriogenology 2012;78:69–76.

[197] Gnemmi G, Lefebvre RC. Bull anatomy and ultrasonography of thereproductive tract. In: DesCoteaux L, Gnemmi G, Colloton J, editors.Practical atlas of ruminant and camelid reproductive ultrasonog-raphy. Ames, IA: Blackwell Publishing; 2010. p. 143–62.