Doppler Ginther

Theriogenology 81 (2014) 112–125
Contents lists available at ScienceDirect
Theriogenology
journal homepage: www.theriojournal.com
40th Anniversary Special Issue
How ultrasound technologies have expanded and revolutionized research

in reproduction in large animals
O.J. Ginther a, b, *
a
Eutheria Foundation, Cross Plains, Wisconsin, USA
b
Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA
a r t i c l e i n f o
Gray-scale ultrasonic imaging (UI) was introduced in 1980 and initially was used to
Article history: examine clinically the reproductive tract of mares. By 1983 in mares and 1984 in heifers/
Received 25 June 2013 cows, UI had become a tool for basic research. In each species, transrectal gray-scale UI has
Received in revised form 6 September 2013 been used extensively to characterize follicle dynamics and investigate the gonadotropic
Accepted 7 September 2013 control and hormonal role of the follicles. However, the use of transrectal UI has also
disclosed and characterized many other aspects of reproduction in each species, including
Keywords: (1) endometrial echotexture as a biological indicator of circulating estradiol concentra-
Arterial pulse tions, (2) relative location of the genital tubercle for fetal gender diagnosis by Days 50 to
Color-Doppler imaging 60, and (3) timing of follicle evacuation during ovulation. Discoveries in mares include (1)
Doppler ultrasonography
embryo mobility wherein the spherical conceptus (6–16 mm) travels to all parts of the
Gray-scale imaging
uterus on Days 11 to 15, (2) how one embryo of a twin set eliminates the other without
Vascular perfusion
self-inflicted damage, and (3) serration of the granulosum of the preovulatory follicle
opposite to the future rupture site as an indicator of imminent ovulation. Studies with
color-Doppler UI have shown that vascular perfusion of the endometrium follows the
equine embryo back and forth between uterine horns and follows the expansion of the
bovine allantochorion throughout each horn. In heifers, blood flow in the CL increases
during the ascending portion of an individual pulse of PGF2a metabolite and then de-
creases. These examples highlight the power of UI in reproduction research. Without UI, it
is likely that these and many other findings would still be unknown.
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction timeline and characteristics of follicle evacuation during

ovulation).
“I wish I could see what’s going on in there,” was a lament This review discusses the early history and eventual lofty
of farm animal reproductive biologists and clinicians status of ultrasound technologies in reproduction research.
throughout most of the 20th century. During the 1980s, Mares and, a few years later, heifers/cows provided the
gray-scale (B-mode) ultrasonic imaging (UI) of the repro- incentive for the initial adaptation and enthusiasm for gray-
ductive organs in horses and cattle revealed dynamic yet scale UI. The premier event (as in Columbus discovering the
unexpected events (e.g., intrauterine travels of the equine New World) was a report in Theriogenology in 1980 by
embryo, acrobatic feats of the equine and bovine fetus). Palmer and Driancourt [1] on UI of the reproductive organs
Before the availability of UI, such activities were not on the of the mare. The report was published a century after the
researcher’s menu or could not be studied without worrying discovery of the piezoelectric effect of certain crystals [2].
about invasive interference. The physical nature of known Crystals in ultrasound transducers have the piezoelectric
events could be contemplated but not described (e.g., characteristic of expanding and contracting in response to
alternating polarities of electric signals. The crystals in the
* Corresponding author. Tel.: þ1 608 798 3777; fax: þ1 608 798 3722. transducer produce ultrasound waves when applied to tis-
E-mail address: [email protected]. sue and receive and convert the resulting echoes back into
0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.theriogenology.2013.09.007
O.J. Ginther / Theriogenology 81 (2014) 112–125 113
electric signals. The signals are converted and displayed as genitalia. Color-Doppler UI and Doppler ultrasonography
shades of gray that represent the intensity of the echoes and are also discussed. The term ultrasonography refers exclu-
the locations of the tissue reflectors. Sounds simple. sively to the production of Doppler graphs that represent
Many research findings in large animal reproduction the changing blood velocities within an arterial pulse and
during the past 30 years can be credited entirely to gray- therefore will be distinct from color Doppler UI.
scale UI. The literature has become so voluminous on Applied and diagnostic uses of UI in reproductive
horses and cattle that some of the sections in this review management in horses, cattle, and other large domestic
have been reduced to a selected list of ultrasound-based species [3–12] and the principles, equipment, and tech-
research contributions that illustrate the research power niques [2,4,13] have been well-reported and are not
of ultrasound technologies in reproductive biology; the considered. The review updates and expands on previous
lists are not comprehensive. In addition, preference is given reviews on research applications of gray-scale UI in horses
to the apparent original reports for historical purposes. The and cattle [2,3,6,14,15]. This review is part of the 40-year
review heralds examples of the capability of UI for data commemorative issue of Theriogenology and notes the
collection and statistical testing of hypotheses by re- role of the journal in disseminating the initial (Table 1) and
searchers and as a source of enlightenment and fascination mounting reports on UI and ultrasonography.
for all who watch the moving images. It is also entertaining
to see something for the first time, even when it is an 2. Gray-scale UI in mares
illusion (Fig. 1). Equine embryo mobility is featured as an
example of a discovery that was solely a product of trans- In the early 1980s, there was a rapid increase in trans-
rectal gray-scale UI. The discovery can be considered rectal gray-scale UI by equine theriogenologists [16–19].
fundamental in that it extended into unexpected areas. Horse-breeding farms were a natural site for initiation of
Consideration will also be given to research findings by the technology for several reasons: (1) intensive veterinary
gray-scale UI in females of large species other than horses management of reproduction in individual mares, (2)
and cattle, specialized research techniques, handling of justification for purchase of relatively expensive equip-
research data, and the application of UI to research in male ment, and (3) fluid-filled embryonal and fetal conceptus
and the noncoiled shape of the uterus permitted intriguing
displays of the dynamic interactions between the conceptus
and uterus. The transrectal route for insertion of the ultra-
sound transducer in the early years came naturally because
of the well-established transrectal route for routine tactile
examination of the internal reproductive organs in mares.
The linear intrarectal transducers that were used for
mares in 1980 to 1982 were of low frequency (e.g., 3.0
MHz). The low resolution of the original transducers is
apparent in published Polaroid ultrasonograms of equine
embryos from Days 11 to 48 [20]. In 1983, a 5.0-MHz linear
transducer was introduced [13]. The 5.0-MHz transducer
had less depth of penetration, but the greater resolution
yielded detailed information on structures close to the
Table 1
Apparent milestones in the introduction of ultrasound technologies for
research study of the reproductive organs in large animals.a
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]
Fig. 1. Examples of image illusions that serve to enliven discussion among
2002 Color-Doppler UI of ovaries in cows [169]
observers. (A) Cross sections of the rear hooves of a 200-day equine fetus
2004 Color-Doppler UI of ovaries in mares [171,174]
and 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 a Gray-scale UI, except for 2002 and 2004.
portion of the fetus. (C) The agonizing ghostly face is an equine CL. (D) The Abbreviations: UI, ultrasonic imaging.
a
arrows delineate a cross section of the pelvic urethra of a stallion during Some of these research milestones were preceded by reports on the
ejaculation. Dilations of the vesicular-gland ducts (eyes) and urethral lumen clinical use of ultrasound for pregnancy diagnoses in sheep, goats, and
(mouth) occur rhythmically with each ejaculatory pulse. Adapted from [2,3] pigs (reviewed in [12]).
b
with permission. References in italics indicate publication in Theriogenology.
114 O.J. Ginther / Theriogenology 81 (2014) 112–125
transducer and separated only by the wall of the rectum. between twins during embryo mobility [27]; (6) a tech-
Consequently, published ultrasonograms of the echo- nique for manual elimination of one member of a twin set
texture of reproductive structures of mares were superior during embryo mobility [28]; (7) physical nature of em-
to those that were available for women, who at the time bryonic death [29]; and (8) postfixation mechanism for
were being scanned through the abdominal wall [2]. Sub- spontaneous embryo reduction of one member of a twin
sequently, this disadvantage was negated with the adap- set (Fig. 3) [30]. The knowledge windfall culminated in the
tation of transvaginal transducers for women. The first text and reference book on gray-scale UI in animals in
ultrasound scanners used in the early 1980s had analog 1986 under the narrow title of “Ultrasonic Imaging and
scan converters; therefore, filming the scans was not a Reproductive Events in the Mare” [31]. It was the most
simple option [2]. By 1986, digital scan converters became exciting 3 years of my ongoing 50 years of research.
available and recording the scans and assessing the scans Ultrasound-based contributions in mares after the 1986
by computerized image analyses increased the research book included many studies on ovarian follicles. After the
power of UI. initial characterization of follicle population dynamics
Equine reproductive physiologists were alerted to the based on diameter groupings [21], the technique of iden-
tremendous potential of gray-scale UI for their own studies tifying individual follicles from examination to examina-
by the original publication in 1980 [1] but were frustrated tion was introduced by researchers in New York [32].
by equipment cost. Our laboratory was fortunate to be Monitoring follicles became essential for research on the
given the loan of a high-quality ultrasound scanner with an hormonal mechanisms associated with follicular waves,
analog converter in 1982. The scanner screen became a selection of a dominant follicle, and hormonal preparation
window to knowledge for our laboratory. Approximately 35 of the ovulatory follicle [33–35]. Follicle dynamics were
publications (63% in Theriogenology) from 1983 to 1985 characterized for the prepubertal stage [36], ovulatory
based entirely on gray-scale UI contributed the following season [35,37], anovulatory season [38], during old age
examples of original research findings: (1) UI dynamics of [39], and in comparison of miniature ponies and larger
the follicles and CL throughout the estrous cycle [21]; (2) UI breeds [40]. It was also found that the characteristics of the
anatomy and pathology of the uterus [22]; (3) echotexture ovulatory follicular wave are surprisingly similar between
of the endometrial folds as an indicator of estrogen expo- mares and women, except for the 2.2-fold larger follicle in
sure for detecting the preovulatory period (Fig. 2) [22,23]; mares at specific events during a wave [41,42]. A major
(4) traveling of the spherical (6–16 mm) embryonic vesicle advance was the finding that both minor waves (waves
to all parts of the uterus (embryo mobility) on Days 11 to 14 without a dominant follicle) and major waves occur in
or 15 (Day 0 ¼ ovulation) [24–26] and fixation on Day 15 or mares [43], and the emergence of each wave type is
16 [19] in ponies and horses, respectively; (5) interactions attributable to a surge in FSH concentration [44].
Original ultrasound-based findings in mares that did not
involve follicular waves after the 1986 book included: (1) first
noninvasive characterization and quantification of uterine
contractions in any species [45]; (2) factors affecting contrac-
tility and role of uterine contractions in embryo mobility [26];
(3) first noninvasive continuous characterization and timing of
Fig. 3. Process of embryo reduction in twin equine embryos with fixation in

the same uterine horn. On Day 17, the thick vascular wall (yellow line) of the
yolk sac of the doomed vesicle (Y2) is forced by uterine massage into the
thin nonvascular wall of the yolk sac of the thriving vesicle (Y1); the vascular
Fig. 2. Contrast between estrus and diestrus in the echotexture of uterine area (red line) of Y1 is shown in contact with the endometrium. Similarly, on
horns in mares (cross sections) and heifers (longitudinal sections). The im- Day 29 the vascular wall of the doomed vesicle (yellow line) is blocked from
ages in heifers are from the curvature at the cranial portion of a horn; the the endometrium. Allantoic sac (A1), yolk sac (Y1, Y2), and embryo proper
arrows delineate the mesometrial attachment. Adapted from [3,6] with (E1, E2) are indicated. 1 ¼ thriving vesicle, 2 ¼ doomed vesicle. Adapted from
permission. [65] with permission.
follicle evacuation during ovulation in any species [46]; (4)

development of fetal gender diagnosis on Days 50 to 60 by
relative location of the genital tubercle (Fig. 4) [47]; (5)
development of the deprivation concept, whereby an embryo
(before Day 40) can eliminate its twin and not suffer self-
inflicted damage [48]; (6) changes in echotexture, especially
that an anechoic layer of the follicle wall distinguishes the
future dominant follicle before the diameter manifestation of
follicle selection (deviation) [49]; (7) discovery of serration of
the granulosa layer of the preovulatory follicle and its use for
predicting imminent ovulation [50]; (8) incidence and nature
of embryonic death, including expulsion through the cervix
[29,51]; (9) characterization of the natural outcome of fetal
twins and in vivo identification of fetal twins in late gestation
by transrectal UI of the apposition of twin fetal membranes
[52]; (10) effects of ultrasonically detected uterine pathology
(e.g., uterine cysts, small intraluminal fluid collections) on
fertility [53] and the conclusion that a small fluid collection
(diameter, 5–20 mm) during diestrus and detectable only by
UI is an indicator of metritis [29,53]; (11) incidence and nature
of dysorientation of the embryo proper and spontaneous
correction [54]; (12) accumulation of fluid in the infundib-
ulum during the estrous cycle [55]; (13) effects of age on
uterine function [56]; (14) assessment of fetal fluids, placental
thickness [57], and ascending placentitis [58]; and (15) dis- Fig. 5. Examples of fetal mobility in a mare and heifer. The equine fetus is
shown with head (HD) and chest (CH) on the allantoic floor and 4 s later
covery that fetal (after Day 40) activity (Fig. 5) involved
being propelled into the allantoic fluid by a vigorous neck (NK) arch. The
frequent changes in orientation and in location among the amniotic membrane is indicated by arrows. The bovine fetus has done a flip-
uterine horns and body, allantoic fluid shifts throughout the flop from side to side as indicated by the rumen (RU) changing from the
uterus, closing and opening of parts or all of each uterine horn, upper to lower position. Adapted from [3,6] with permission.
and closure of both horns at mid-pregnancy with both fetal
hind limbs encased by the umbilical cord horn [59–61].
findings by UI account for the availability of techniques that
Excluding the many research reports involving follicle dy-
can be used by equine clinicians. These are indicated above by
namics, 24 findings or characterizations in mare reproductive
points 3 and 6 for 1983 through 1985 and points 4, 7, 9, and 10
physiology are listed that likely would still be unknown or
during and after 1986.
unclarified if it were not for the noninvasive but powerful
ultrasound technology of transrectal gray-scale UI. These
findings and techniques give credence to the opinion enun- 3. Equine embryo mobility
ciated in the title of this review. In addition to monitoring
follicles and detecting ovulation, nonfollicular research Some of the discoveries in mares that are attributable to
transrectal UI can be considered fundamental. For example,
the demonstration of equine embryo mobility (Fig. 6),
wherein the embryo travels to all parts of the uterus an
equivalent of 10 to 20 times per day on Days 11 to 15 [25,26]
was a stark departure from the assumption that the embryo
at this time was nestled snugly among the endometrial
folds. As the encapsulated embryo moves, it carries mes-
sages for interaction with the uterine wall (Fig. 6). One
message stimulates myometrial contractions, which pro-
vide the propulsive force for the embryo [26,62], and
another stimulates an increase in vascular perfusion in the
uterine wall that moves approximately every 0.5 hour from
horn to horn with the embryo [63]. The essentiality of
embryo mobility involves a message that is carried to all
parts of the uterus to block the uterine luteolytic mecha-
nism, ensuring the required maintenance of the
progesterone-producing CL [64]. All of the uterus must be
visited by the relatively small embryo because in this spe-
Fig. 4. Frontal views across the front limbs (FL), hind limbs (HL), umbilical cies the luteolytic signal from uterus to CL is through sys-
cord (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 fetus
temic channels [65]. On the days of mobility, a substance is
and caudal to the hind limbs in the female fetus. Adapted from [6] with distributed that gradually stimulates increased uterine tone
permission. [62,66]. By the time the blockage of luteolysis is complete
report from France on pregnancy diagnosis in 1982 [70] and

reports on the echotexture of the reproductive organs and
conceptus appeared in Theriogenology in 1984 [71–73].
Apparently, the greatest current use of UI in cattle in most
laboratories is to determine the time of ovulation as a
reference point or end point and to diagnose pregnancy.
However, the technology has many more uses and poten-
tial. Detailed studies have been done on follicle dynamics
before puberty [74], during the estrous cycle [75], during
early [76] and late pregnancy [77], and during super-
stimulation [73]. The technology has become essential for
collection of follicle data in cattle, as for mares, especially
by maintaining the identity of follicles from examination to
examination [78]. The follicles have been studied during a
wide array of research projects involving many aspects of
follicle dynamics, waves, and selection [33,79]. Studies with
follicles have included the demonstrations that the emer-
gence of each follicular wave during the estrous cycle [80]
and throughout pregnancy [77] is associated with a surge
in FSH and that a close two-way coupling is involved in the
follicle/FSH relationship [81,82]. A recent study used
transrectal UI of follicles to study roles and controls of the
preovulatory and periovulatory FSH surges [83]. Echo-
texture reflects the function and endocrine status for the
Fig. 6. Sequence (1–4) of events associated with equine embryo mobility,
follicular antrum and wall and for the CL [84–86]. Charac-
uterine contractions, blockage of luteolysis during the first luteal response to
pregnancy, decrease in uterine diameter from an increase in tone, and fixation
terization of the nature and timing for evacuation of
at a flexure in a caudal uterine horn. Adapted from [162] with permission. approximately 90% of the antral fluid during ovulation
involved 4 s [87], compared with 60 s in mares [46]. Studies
on follicle dynamics and selection have included other
(mean, Day 16), the embryonic vesicle has grown and bovine species such as Bos indicus [88] as well as B taurus;
uterine tone has increased (decreased uterine diameter) to for example, deviation in the first postovulatory wave in
the point that fixation occurs at the flexure in a caudal horn Nelore heifers and cows occurs approximately 1 day earlier
that offers the greatest impediment to continued mobility when the future dominant follicle is 2 mm smaller than in
[26,67]. Holsteins.
Embryo mobility also provided explanation or rationale Research findings in cattle based on transrectal gray-scale
for hypotheses on several phenomena that were perplexing UI have included nonfollicular structures and events as fol-
to equine veterinarians and biologists [68], including: (1) lows: (1) changes in size and echotexture of the CL [73],
lack of agreement between side of ovulation and side of leading to comparisons of functional and structural CL
fixation (embryo mobility involves both horns with no changes [89] and to the use of UI to define a change in CL area
affect of side of CL), (2) preferential fixation in postpartum (cm2) as a reference point for initiating experiments [90]; (2)
mares in the formerly nongravid horn (nongravid horn is ultrasonic appearance of the uterus, including a more het-
smaller), (3) fixation almost always in the caudal portion of erogeneous echotexture during estrus (Fig. 2) [91]; (3)
one of the uterine horns (bend or impediment to mobility reduced curling of the uterine horns during estrus [91], un-
in caudal horn), (4) fixation 1 day earlier in ponies than in like the apparent curling during estrus when based on
horses (uterus is smaller in ponies but embryonic vesicle is transrectal palpation; (4) characterization of uterine con-
not), and (5) greater incidence of unilateral than bilateral tractions during the estrous cycle and pregnancy [92]; (5)
fixation of twins (first embryo to become fixed is an pregnancy characterization [72,93], including detailed de-
impediment to mobility by the other). Ultrasound scanners scriptions of the conceptus on Days 10 to 60 [94–96]; (6)
were the window to the phenomenon of embryo mobility nature of embryonal and fetal loss [97–99] and factors
and the means for characterizing the underlying mecha- involved in the loss [100]; (7) determining fetal sex by rela-
nisms. Furthermore, embryo mobility and fetal activity and tive location of the genital tubercle (Fig. 4) [101] and later by
interactions with the uterus are spectacular events that identification of genitalia [102]; (8) transient filling and
inspire awe and respect for animal biology. With regard to emptying of various segments of the uterus by the fluid-filled
the impact on laymen as well as professionals, a survey by placental membranes, gradual increase in intrinsic activity of
EQUUS magazine credited embryo mobility as an the fetus between Days 40 to 60, and changes in recumbency,
outstanding discovery in equine research [69]. presentation (cranial vs. caudal direction), and intrauterine
locations throughout pregnancy (Fig. 5) [6]; (9) assessing
4. Gray-scale UI in heifers/cows uterine inflammation [103] and postpartum ovarian activity
and uterine involution [104,105]; (10) detection of twins [99]
The first reports on transrectal gray-scale UI for exam- and spontaneous loss [106], and ultrasound-guided reduc-
ining the reproductive tract of heifers/cows appeared in a tion of one member of a twin set [107,108]; and (11)
discovery that several fetal anomalies can be detected by UI was shown to be monovulatory with two follicular waves
on Days 55 to 120, including compact fetal mass, schistoso- during the follicular phase. Recent studies have included
mus reflexus, and fetal hydrops [6]. Early detection (e.g., color Doppler and three-dimensional UI to describe luteal
during fetal gender determination) and termination of the and conceptus development in elephants [128]. In the
pregnancy is an advantage of UI in that many of these original study [126], a nontranquilized rhinoceros was used
anomalies result in dystocia at term. Although bovine re- to identify individual ovarian follicles for 34 days and an
searchers and clinicians were slower than their equine ultrasonogram of a 27-day embryo was recorded. The em-
counterparts to embrace gray-scale UI, the eventual results bryo of the rhinoceros resembled an equine embryo at a
further entrench the accolades in the title of this review. similar stage and was in a similar location at the caudal
uterine horn. Ultrasound-guided oocyte recovery in the
5. Gray-scale UI in other large species rhinoceros has also been reported [129]. The status of gray-
scale UI in the rhinoceros and elephant and the role of UI in
The experiences with horses and cattle encouraged the assisted reproduction have been reviewed [130].
use of transrectal UI for evaluating the reproductive organs in Researchers in Canada have used transrectal UI to study
sheep, goats, pigs, llamas, water buffalo, and nondomestic the ovaries in camels [131] and in several large species of
large animals. The transabdominal route may be used in wildlife; approximately 50% of the reports were in Ther-
smaller species (e.g., sheep), especially for pregnancy diag- iogenology. The ovaries of elk (wapiti) were studied in detail
nosis [2], but the resolution of transrectal UI is needed for for the anovulatory and ovulatory seasons [132–134],
detailed studies. For transrectal UI, a transducer extension or leading to programs for ovarian synchronization, induction
a person with a small arm may be needed for rectal insertion of follicular waves, and superovulation. Superstimulation of
and manipulation of the transducer [2]. An extension may follicles, oocyte collection, embryo transfer, and ovarian
also be used for very large animals (e.g., elephants). In sheep, synchronization programs have also been developed for
UI has been used for diagnosing and studying pregnancy bison after serial study of the ovaries by transrectal UI
[109,110], assessing ovarian structures [108,109], studying [135]. The reproductive organs of moose [136], muskoxen
the association between emergence of follicular waves and [137], and seals [138] were also studied by transrectal gray-
FSH surges [111], and determining follicle dynamics with scale UI.
high and low ovulation rates [112]. Pregnancy and follicular
waves also have been characterized by UI in goats [113,114] 6. Specialized gray-scale UI techniques
and pigs [115,116]. Earlier studies in llamas demonstrated:
(1) echotexture and morphology of the reproductive organs, Ultrasound technologies have become increasingly
including straightening and curling of the uterine horns important not only for viewing and characterizing the
during estrus and diestrus, respectively [117]; (2) follicular images of moving events (e.g., uterine contractions) and
waves, including growth and regression of individual folli- slow events (e.g., follicle growth), but also as a crucial
cles [118]; and (3) echotexture of the CL [119]. Subsequent component or guide for specialized techniques. The listing
studies in Canada in llamas have utilized UI as an aid in of the following techniques is intended as an overview,
transvaginal collection of oocytes [120] and for a series of and preference is given for the reports that apparently
studies that culminated in the first demonstration of an introduced or popularized a technique: (1) first use of
ovulation-inducing factor in the seminal plasma of llamas, ultrasonically simulated biological structures (equine
alpacas, and bulls [121]. Follicular waves have also been embryos) for reproduction research in any species [26];
shown by UI to occur in water buffalo [122,123] and have (2) computerized pixel analyses of ovarian structures in
implications for assisted reproduction [124]. mares [139,140], heifers [84–86,141,142], and ewes [143];
Although slow to be adopted, transrectal scanners are (3) observing images of semen streaming into the uterus
without parallel in the study of the reproductive organs of as an aid for teaching and evaluating AI technicians [144];
nondomesticated large animals. The knowledge gained on (4) first attaching of echogenic markers in reproduction
the reproductive system through transrectal UI and other research in any species (horses [145]); (5) guiding can-
ultrasound techniques (e.g., oocyte collection) have shown nulation into the caudal vena cava for sampling blood with
potential in aiding these species to propagate, especially a greater proportion of ovarian effluent in heifers [146];
those that are endangered. In farmed red deer, transrectal (6) inserting a research substance directly into a follicle in
UI was used in 1990 to establish criteria for predicting mares [147] and heifers [148]; (7) sampling follicular fluid
calving data by measurements of the uterus, amniotic sac, in heifers [149] and mares [150]; (8) sequential biopsy of
placentomes, and several parts of the fetus [125]. The the CL in heifers [151] and mares [152]; (9) aspiration of
introductory publication on transrectal UI for large zoo follicle contents for functional follicle ablation in mares
animals appeared in 1991 and provided descriptions and [153] and heifers [154]; (10) recovering and transferring
ultrasonograms of the internal reproductive tract in Asian oocytes into other follicles by transvaginal UI in cattle
and African elephants, black rhinoceros, white rhinoceros, [155] and mares [156] aided by manual transrectal
banteng, gaur, giraffe, and bactrian camel [126]. In ele- manipulation of an ovary; (11) studying the develop-
phants, ultrasonically detected changes in the cervix and mental patterns of small follicles (1–3 mm) by transrectal
uterus were described. Subsequent studies in Germany UI in cattle [157]; (12) ultrasonic biomicroscopy for in vivo
using transrectal UI have elucidated many of the unusual imaging of surface follicles as small as 0.4 mm (including
aspects of the reproductive cycle of the elephant that were the cumulus–oocyte complex) in cattle, using an ultra-
unknown before UI became available [127]. The elephant sonic biomicroscope with a single crystal that emits
ultrasound waves of 20 to 70 MHz [158]; (13) evaluating scale or color-mode image of the lumen of an artery (Fig. 8).
moving structures such as the fetal heart by M-mode The focused results from the sample gate are displayed on
(motion mode) [2]; (14) transvaginal ultrasound guiding the screen by a graph that represents changing blood flow
for inseminating and transferring embryos directly into a velocities at various times within a cardiac cycle or indi-
uterine horn by bypassing the cervix [159]; and (15) vidual arterial pulse. The numerical velocities are
aspirating oocytes by transvaginal ultrasound scanning computed and displayed on the screen for a selected car-
and guiding for in vitro fertilization [160]. Thus, the use of diac cycle. Doppler indices (resistance index; pulsatility
specialized techniques further enhances the prestige of UI index) are also displayed on the screen. The indices are
as a boon to research. On the applied side, the in vivo re- ratios computed from the various points of the changing
covery of oocytes has become common in the embryo velocities in the cardiac cycle. They are especially useful for
transfer industry subsequent to its publication in Ther- the tortuous arteries of the reproductive tract because they
iogenology by researchers in The Netherlands [160,161]. are independent of the angle of the transducer to the angle
of blood flow. These indices reflect the hemodynamics of
7. Color-Doppler UI and ultrasonography the tissue supplied by the artery distal (downstream) to the
sample gate. An increase in the resistance index or the
Doppler ultrasound is not used as frequently as gray- pulsatility index indicates an increase in resistance and
scale ultrasound and therefore the principles of the therefore a decrease in vascular perfusion of the tissues.
Doppler technology are discussed briefly. The Doppler Techniques for locating an image of a cross-section of the
technology is based on Doppler-shift frequencies, wherein uterine and ovarian arteries in mares [162,164] and heifers/
the frequency of echoes from moving red cells is increased cows [162,164,165] have been described. The locations of
or decreased as the cells move toward or away from the the pelvic arteries to the genitalia of stallions and bulls are
transducer [4,162,163]. The Doppler effect of ultrasound is also illustrated [162].
similar to the Doppler effect of sound, wherein the sound The use of transrectal Doppler ultrasonography for
frequency changes as the source (e.g., car horn) moves to- research studies in large animal reproduction was first re-
ward or away from a listener. Color-Doppler UI and Doppler ported by researchers in Germany in a series of publications
ultrasonography provide distinctively different approaches in Theriogenology, beginning in 1998 in mares [164] and in
for assessing the vascular system of reproductive organs. In 2000 in cows [165]. Ovarian and uterine blood flow ve-
color-flow mode (Doppler UI), the direction of blood flow locities during the estrous cycle and uterine and umbilical
relative to the face of the transducer is represented by blood flow during pregnancy were included. Their studies
different colors on the screen display (Fig. 7). The extent of on blood velocity spectral graphs in cattle during the
color can be estimated by percentage of a tissue with color estrous cycle, pregnancy, and postpartum have been
signals or can be calculated by computer, based on number reviewed [163]. Vascular perfusion of the CL and uterus was
of colored pixels. greater during the first versus second follicular wave of the
In spectral mode (Doppler ultrasonography), blood flow estrous cycle [166]. Color-Doppler displays of blood-flow
for a focused location in a specific artery is assessed by signals in the follicle and CL in cattle have been popularized
placing a sample-gate cursor (e.g., 1 mm wide) on the gray- by researchers in Japan [167–170].
The following research findings and conclusions are
primarily from our laboratory and are used to illustrate the
power of color Doppler UI and ultrasonography in repro-
duction research. Findings in mares include: (1) during
follicle selection, blood flow in the follicle wall begins to
increase in the future dominant follicle compared with the
largest future subordinate follicle an equivalent of 1 day
before diameter deviation [171] and is similar to the day of
increasing prominence of the anechoic band [49]; (2) per-
centage of follicle circumference with blood flow signals
begins to decrease in the follicle wall 4 hours before
ovulation [172]; (3) serration of the granulosa opposite to
the future site of ovulation results from blood vessels
beneath the granulosa [173]; (4) blood flow in the wall of
the dominant follicle (>30 mm) is less in anovulatory fol-
Fig. 7. Ultrasonograms from gray-scale UI and color Doppler UI of a longi- licles than in ovulatory follicles during the transition be-
tudinal section of the spermatic cord of a bull. The gray-scale image shows tween anovulatory and ovulatory seasons [174]; (5)
anechoic sections of the highly convoluted testicular artery as it intertwines
percentage of wall of the preovulatory follicle with blood
with the network of veins of the pampiniform plexus. Some of the arterial
sections are rounded (arrows) with specular reflections on the upper and flow signals is greater in mares that subsequently become
lower surfaces, indicating a smooth arterial wall and a perpendicular di- pregnant than for those that do not [175]; (6) percentage of
rection of the ultrasound beams. The color-Doppler image shows arterial the circumference of a preovulatory follicle with blood flow
blood in red or blue depending on the direction of blood flow relative to the signals is greater and includes the apical area (site of po-
ultrasound beams. Arterial cross sections that are devoid of color (arrows)
indicate that the ultrasound beams intersected the blood flow at a 90 angle
tential rupture) when a hemorrhagic anovulatory follicle
so that the direction of flow was neither away nor toward the transducer. forms than when the follicle is ovulatory [176]; (7) neither
Adapted from [162] with permission. an 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 focus
that will be the source of the graph of velocities in the selected cardiac cycle. The angle cursor (AC) is placed by operator to indicate the angle of blood flow to the
plane of the ultrasound beams. Blood velocities of the systolic and diastolic portions of the cardiac cycle are depicted. Blood velocity is determined and listed on
the scanner screen at various points of the velocity graph or spectrum. In this example, the average velocity is shown and is called time-averaged maximum
velocity (TAMV). The resistance index is also shown. A greater index indicates less blood flow distal to the sample gate. Adapted from [162] with permission.
before the beginning of a progesterone decrease in spon- ultrasound technologies [63]. Vascular perfusion was
taneous luteolysis [177]; (8) after follicle evacuation, assessed by the estimated score for number of color signals
vascularization of the CL begins at the basal area (site of in a section of the endometrium, by computer-generated
granulosa serration in the preovulatory follicle) and pro- number of color pixels (color-Doppler UI), and by blood
gressively extends toward the apical area during Days 0 to 6 velocity in various parts of an arterial pulse in an artery in
[173]; (9) color-Doppler signals apparently in the endo- the mesometrial attachment (Doppler ultrasonography).
metrium mark the future post-orientation site of the Each of the three approaches indicated similarity between
equine embryo proper well before the embryo proper is the nonpregnant and pregnant mares until Day 12. On Day
detectable by gray-scale imaging [178]; (10) heart rate of an 13 of pregnancy, vascular perfusion by estimation of the
equine embryo on Days 24 and 27 was lower by more than percentage of color signals or by computerized pixel count
3 standard deviations in an embryo that was lost on Day 31 was greater in the uterine horn with the mobile embryo.
than in controls [162]; (11) reduced uterine vascular After fixation (end of embryo mobility), each of the two
perfusion is associated with uterine cysts [179]; and (12) assessments by color-Doppler UI and a lower pulsatility
the movement of the conceptus back and forth between index in the assessment by ultrasonography indicated
uterine horns during embryo mobility is accompanied by a greater vascular perfusion in the horn of fixation than in
back-and-forth transient increase in vascular perfusion of the opposite horn.
the endometrium [63], and at the end of mobility perfusion The heart beat of the equine embryo is detectable on
is greater in the horn of fixation [63,180]. Days 17 to 20 or approximately 2 days earlier by color-
Color-Doppler UI research findings in heifers/cows Doppler UI than for detection of the embryo proper by
include (1) the percentage of follicle wall with blood flow gray-scale UI [162]. Color-flow signals extend beyond the
signals increases synchronously with the initiation of the LH heart and into vessels of the membranes by Days 27 or 30.
surge [168]; (2) the percentage of wall of the preovulatory In cattle, color-flow signals extend into the vessels by Day
follicle with blood flow signals is greater in heifers that 28 and are detectable in the umbilical cord, aorta, and ca-
subsequently become pregnant [181]; (3) during luteolysis, rotid artery. Color Doppler images of the embryo (before
blood flow in the CL increases during the ascending portion Day 40) and fetus (after Day 40) are shown for horses and
of each pulse of PGF2a metabolite, remains elevated for 2 cattle (Fig. 9). Extensive series of color-Doppler images for
hours and then decreases [182]; (4) the increased CL blood the follicles, CL, uterus, embryo, and fetus in horses and
flow during PGF2a treatment is from the direct stimulation cattle and examples of determination of blood velocities
of PGF2a on the CL vasculature in heifers [183]; and (5) and resistance indices by ultrasonography have been pub-
endometrial scores for an increase in the extent of vascular lished [162].
perfusion follow expansion of the allantochorion thr-
oughout each uterine horn in cattle [184]. These examples of 8. Handling of research data
research findings in mares and heifers highlight the capa-
bilities of UI in reproduction research and would not have Despite the more than 30-year history of ultrasonic
been possible without the availability of transrectal color- technologies as research tools, there seems to be a reluc-
Doppler UI. A text and reference book with colored images tance to apply statistical analyses to data obtained by gray-
and more detailed discussion has been published [162]. scale or color-Doppler UI, except for data from studies of
A study on vascular perfusion of the endometrium in follicles and follicular waves. Many other quantitative end
pregnant mares is used to illustrate the approaches that can points are available through the imaging technologies
be used in uterine blood flow studies by the Doppler [2,3,6] as indicated by the examples in previous sections.
Fig. 9. Color-Doppler ultrasonograms of the embryonic vesicle and fetus in a mare and in a heifer. as, allantoic sac; ao, aorta; ca, carotid artery; dv, ductus
venosus; ep, embryo proper; fl, front limb; ht, heart; hv, hepatic vein; hl, hind limb; jv, jugular vein; mm, mesometrial attachment; uma, umbilical artery; umv,
umbilical vein; vc, vena cava; ys, yolk sac. Adapted from [162] with permission.
End points that can be quantified and compared among concentrations. The scoring system is subjective in that the
groups include: (1) diameter or other dimensions of the operator’s judgment is used. Bias can be minimized by
embryonic vesicle, embryo proper, and fetus and other filming the images so that scoring can be done without
measurable structures involving the conceptus; (2) diam- knowledge of source, or scoring can be done by a second
eter, area, or volume of the CL; (3) embryo and fetal heart
rate; (4) extent of embryo mobility measured by distance
and time in mares; (5) diameter or other measures of the
uterus (Fig. 10); (6) height or other measures of fluid
pockets in the uterus; and (7) percentage of a structure
(e.g., CL) with color pixels, indicating the extent of vascular
perfusion. With regard to follicles, diameter is a convenient
measure that is readily understood. However, there may be
instances when surface area (as calculated from a cursor
tracing of the periphery) may be more meaningful because
function is most related to the periphery of the antrum [2].
Irregular structures (e.g., equine CL) can be traced for area
(cm2) determination. Many scanners have convenient ca-
pabilities for obtaining such measurements and displaying
the numerical results on the screen. Thus, ultrasound
quantitative data can be used for statistical comparisons
among time or groups similar, for example, to data for
changes in hormone concentrations.
Scoring of end points from minimal to maximal (e.g., 1–
4) is a useful technique for image characteristics that are
not amenable to quantifying by conventional measuring
techniques (Fig. 10) [2]. Scoring can be useful, for example,
for relative amounts that cannot be easily measured, such
as intrauterine fluid, endometrial edema, extent of uterine
contractions, changes in uterine shape, and percentage
estimate of a defined area with color signals. The scoring
approach facilitates profiling and statistically analyzing
Fig. 10. Examples of a quantitative measurement (height from dorsal to
changes and analyzing temporal relationships or correla- ventral surface) and a subjective score (0 [nil] to 4 [maximum]) for a bovine
tions among the ultrasonically monitored events as well as uterus. Each end point can be subjected to statistical analyses for differences
among other events, such as changes in hormone among days. Adapted from [6] with permission.
operator who is unaware of the source or even the hy- 9. The male
pothesis under test. We have developed considerable
confidence in the scoring approach in our laboratory Stallions and bulls have not been neglected in the utili-
because of consistent results among experiments and in- zation of transrectal and transcutaneous gray-scale and color
dependent operators and the ease of analyzing and inter- Doppler UI [3,6,162]. Transrectal gray-scale UI apparently
preting results in relationship to known biological was first used in stallions in 1987 in a description of the
mechanisms. accessory sex glands [186]. Researchers in Idaho described
Information from gray-scale and color-Doppler UI can be the changes in the accessory sex glands before and after
digitized and analyzed by computer. Digitization is a proce- sexual preparation (teasing of mares) [187]. The vesicular
dure in which each pixel of an image is assigned a numerical glands became rounded after sexual preparation from the
value for brightness in gray-scale UI, or the number of colored entry of vesicular gel, as indicated by an increased anechoic
pixels is counted in color-Doppler UI [2]. Conversion of image area. The Idaho researchers also developed a unique system
information to numerical data allows the use of quantitative for transrectal UI of the sex glands during ejaculation
statistics for temporal characterizations or testing of hypoth- [188,189]. Apparently for the first time in any species, images
eses. However, the sophisticated processing does not elimi- of the dynamics of the sex glands were observed in real time.
nate bias. The operator retains the responsibility of selecting Rhythmic pulses (emissions) of the prostate secretions
and delineating the areas to be processed, and these decisions occurred before the onset of the manually determined penile
are notoriously subject to bias. Computer pixel analyses, and urethral rhythmic contractions or sequential ejaculatory
therefore, can be dangerous and lull the operator into a false pulses. Discharge of the prostate continued during the first
sense of confidence. It is best if the operator selecting the few ejaculatory pulses. Rhythmic ampullary and prostatic
images or delineating the area for study does not know the activity began a few seconds before the start of ejaculation.
experimental group or source of the image. Pixel analyses Ampullary fluid passed through the ducts in distinct boluses
have been used, for example, to characterize day-to-day before ejaculation, and prostate emission continued for the
changes in the intensity or echogenicity of the CL [86,140], first few ejaculatory contractions. In contrast, the emission of
quantify changes in echotexture [85] and shape changes in the vesicular glands with accompanying pulses in the ducts
preovulatory follicle as ovulation approaches [139], and occurred after ejaculatory or urethral contractions were
evaluate the extent of vascular perfusion [4,162]. Detailed underway (Fig. 1). These ultrasonically determined patterns
information on statistical handling of ultrasonic images and of emission are consistent with the order of appearance of
pixel analyses in cattle [6] and horses [3] is available. the various portions of the seminal fluid in stallion ejacu-
The images displayed on the ultrasound screen repre- lates. The results demonstrated the power of UI in physically
sent interactions between the technology and living tissues active organs.
[2]. Artifactual and factual echoes are produced, and The technique for transcutaneous imaging of the
consequently, embarrassing errors may be published. external genitalia in stallions has been described [190]. A
Proper instrument adjustment and proper interpretation of standoff pad is used on the ventral surface of the testes for
the echoes on the ultrasound screen are crucial. Interpre- imaging the branches of the testicular artery. The central
tation requires knowledge of the relationships between vein can be imaged running longitudinally. Abnormalities
tissues and echoes and the ability to differentiate between (e.g., tumors, focal inflammation) can be imaged. Abnor-
true and artifactual responses. There are two types of gray- malities of the epididymis and the cause of scrotal
scale reflections (specular and nonspecular) that are pre- enlargement (e.g., gravitated fluid, inguinal hernia, blood
sented as echoes [2]. Artifactual echoes can be mistaken for clot) also may be detected by transcutaneous UI.
echoes that represent actual structures. In addition to The genitals of bulls have also been examined by gray-
specular echoes, certain tissue formations also cause ul- scale UI. The examinations are effective for assessment of
trasonic waves to bend (refract), bounce back and forth or structure and for generating research data [6,191]. Reports
re-echo (reverberate), become weakened (attenuated), on the initial imaging of the bull testis in vitro in 1987 [192]
entirely blocked (shadowing), or exaggerated (enhanced). and in vivo in 1988 [193] and the ultrasonic appearance of
As a result, distortions appear on the ultrasonic image, the accessory sex glands [191] were first published in
which can be mistaken for normal or pathologic structures Theriogenology. Mean pixel intensities from ultrasonograms
or changes. Artifacts are especially common during UI of of testes parenchyma indicate that gray-scale UI may be a
the reproductive tract because of the many pockets of useful noninvasive method for determining reproductive
bowel gas, fluid-filled structures, and the pelvic bone. development in maturing bulls [194]. The accessory sex
Artifacts are a nuisance in color-Doppler work [162]. glands are imaged transrectally and the testes, epididy-
Clutter artifacts for Doppler UI and distorted graphs of the mides, and the testicular cones, transcutaneously. The
cardiac cycle in Doppler ultrasonography are most common vascular cone consists of the highly coiled testicular artery
in transrectal examinations and result from movements of (Fig. 7) intertwining with the venous network the pampi-
tissues, the animal, or transducer. Animal restraint or niform plexus. Recent reports concluded with the opinion
acclimation and filter settings can be used to lessen the that the primary use of UI in the assessment of reproductive
clutter. In this regard, detomidine sedation in horses and function in bulls is the detection of lesions in the testes and
xylazine sedation in heifers affects blood flow in major scrotum [195], but assessing breeding soundness is limited
arteries (e.g., internal iliac) but an effect on local vascular [196]. Recent reviews are available for the many publica-
perfusion in the ovaries and endometrium has not been tions on monitoring reproductive function by UI in bulls
detected [185]. [196,197].
10. Conclusions production in the 21st century. Saskatoon, Saskatchewan, Canada:

University of Saskatchewan; 1994. p. 133–45.
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There can be little doubt that transrectal gray-scale UI,
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Acknowledgments [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.
Production of this manuscript was supported by the Proceedings of Am Assoc Equine Pract, 1983:11–26.
Eutheria Foundation, Cross Plains, Wisconsin. The author [29] Ginther OJ, Garcia MC, Bergfelt DR, Leith GS, Scraba ST. Embryonic
loss in mares: pregnancy rate, length of interovulatory intervals,
thanks John Kastelic for critiquing a presubmitted copy of
and progesterone concentrations associated with loss during days
the manuscript and Maria Hoffman for word processing 11 to 15. Theriogenology 1985;24:409–17.
and figure preparation. [30] Ginther OJ. Postfixation embryo reduction in unilateral and bilat-
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[31] Ginther OJ. Ultrasonic imaging and reproductive events in the
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Theriogenology 81 (2014) 112–125

Contents lists available at ScienceDirect

40th Anniversary Special Issue

How ultrasound technologies have expanded and revolutionized research

1. Introduction timeline and characteristics of follicle evacuation during

Year Ultrasound technologyb

Fig. 3. Process of embryo reduction in twin equine embryos with ﬁxation in

follicle evacuation during ovulation in any species [46]; (4)

report from France on pregnancy diagnosis in 1982 [70] and

10. Conclusions production in the 21st century. Saskatoon, Saskatchewan, Canada:

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