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Research Article |
Eutheria Foundation, Cross Plains, Wisconsin 53528
ABSTRACT
The spherical equine embryonic vesicle is mobile throughout the uterine lumen for several days before becoming fixed in the caudal segment of a uterine horn on Day 16 (ovulation = Day 0). Orientation refers to the position of the embryo proper at the periphery of the vesicle relative to the position of the mesometrial attachment. In mares, the embryonic pole of the vesicle is antimesometrial after completion of orientation. Day of vesicle fixation, differential thickening of the endometrium near the mesometrial attachment, and orientation of the embryonic vesicle were studied in 30 ponies, using B-mode and color-Doppler transrectal ultrasonography. The thickness of the endometrium at the mesometrial aspect of the vesicle divided by the thickness at the antimesometrial aspect was termed the encroachment ratio. At the future site of fixation, the first increase (P < 0.05) in the encroachment ratio occurred between 4 and 1 days before fixation. An early vascular indicator of the future position of the embryo proper was discovered by color-Doppler imaging and consisted of a colored spot in the image of the endometrium close to the wall of the embryonic pole. The early indicator was detected in each mare 0.5 ± 0.1 days after fixation and 2.5 ± 0.2 days before first detection of the embryo proper. The position of the early indicator when first detected at the periphery of the embryonic vesicle was not significantly different from the position of the embryo proper when first detected. Results supported the hypothesis that differential thickening of the endometrium precedes orientation and indicated that orientation occurs immediately after fixation.
color-Doppler, conceptus, embryo, endometrium, female reproductive tract, mares, orientation, pregnancy, uterus
INTRODUCTION
Species of the genus Equus apparently are the only common eutherian mammals in which embryo-uterine interactions (migration, fixation, and orientation) can be observed in real time and apparently without disturbance. This research capability results from the availability of transrectal ultrasonography, the large size of the fluid-filled embryonic vesicle (conceptus), and the close proximity of the uterine wall to the rectal wall [1].
The equine embryonic vesicle enters a uterine horn on Day 6 (ovulation = Day 0 [2, 3]). Based on a uterine-ligation experiment [4], the vesicle enters the uterine body on about Day 8. When first detected by ultrasound (Days 9–10), the vesicle is a distinctly spherical 3- to 4-mm structure and is usually (60% of the time) found in the uterine body [5]. Thereafter, mobility of the vesicle increases, and the vesicle reaches extensive mobility on Day 11. During the extensive mobility phase, the vesicle traverses the full length of the uterus many times per day [6]. Each of the uterine horns and the uterine body are similar in length, and the mobile vesicle spends a similar amount of time in each part. Embryo mobility is associated with uterine contractions [7]. A recent study indicated that transient changes in endometrial vascular perfusion accompany the location changes of the embryonic vesicle [8]. Fixation (cessation of mobility) occurs at a flexure in the caudal segment of a uterine horn under the influence of increasing growth of the vesicle and a reciprocal relationship between increasing uterine tone and decreasing uterine diameter [9]. Vascular perfusion is greater in the endometrium surrounding the fixed vesicle than in the opposite horn or in the middle of the horn of fixation [8].
Orientation of the embryonic vesicle refers to the position of the embryonic disc or embryo proper at the periphery of the vesicle (embryonic pole) relative to the position of the mesometrial attachment. The pattern of orientation (antimesometrial versus mesometrial) is fairly constant within species but differs among species [10, 11]. The direction in which the embryonic disk faces relative to the mesometrial attachment after orientation is completed contributes to species differences in the pattern of development of the fetal membranes and the site of attachment of the umbilical cord. When first detected by ultrasound (Days 19–22), the equine embryo proper is in the ventral hemisphere of the embryonic vesicle or opposite to the mesometrial attachment [12]. It is unlikely that orientation occurs before embryo mobility ceases. In this regard, simulated embryonic vesicles rotated or rolled during intrauterine location changes [13]. These observations indicate that orientation occurs between the day of fixation (Day 16) and the earliest reported day of ultrasonic identification of the embryo proper (Day 19).
About 50%–75% of the wall of the equine embryonic vesicle over Days 16–18 is composed of two cell layers without mesoderm [14]. The mesoderm of the remaining portion develops between the two cell layers and differentiates into connective tissue surrounding the embryonic disc, resulting in a three-layered portion of the vesicle wall at the embryonic pole. Thus, the three-layered or embryonic pole of the vesicle can be expected to have greater tensile strength than the opposite pole, although this has not been determined directly. Beginning on approximately Day 17, the embryonic vesicle begins to lose its spherical form when imaged in cross-section relative to the uterine horn. The ultrasound image of the vesicle becomes oblong, triangular, or irregular in shape [12]. The apex of the triangular shapes tends to be at the dorsal region of the horn. However, the shape of the vesicle does not remain static, and its outline changes frequently during periods of continuous ultrasound observations with the scanner [14]. These shape changes are attributable to myometrial contractions, which may exert a kneading or massage-like action on the fixed vesicle. Disproportional thickening of the dorsal uterine wall versus the ventral wall occurs by Day 17 and accounts for the nonspherical shapes of the vesicle [12].
It has been postulated [12, 13] that equine embryo orientation results from the interaction of at least three factors: 1) differences in tensile strength between the thin (two cell layers) and thick (three layers) portions of the vesicle wall; 2) asymmetrical encroachment of the uterine wall on the vesicle, resulting from differential thickening of the upper turgid uterine wall at the mesometrial attachment; and 3) the massaging action of uterine contractions. A distinct, smooth, and strong capsule encloses the embryonic vesicle until about Day 21 [15] and is an additional factor that likely favors the orientation process. The surface of the equine embryonic vesicle develops adhesive qualities [16] that may aid in anchoring the vesicle after orientation is completed.
The overall purpose of this experiment was to examine the hypothesis that differential thickening of the uterine wall at the mesometrial attachment begins before the earliest indication that orientation has occurred. Initially, a search was made for early indicators of the position of the embryonic disc at the periphery of the vesicle, using color-Doppler assessment of the endometrium and vesicle (Experiment 1). The time of the occurrence of differential thickening of the dorsal uterine wall at the site of fixation was then examined (Experiment 2). Findings were considered supportive of the hypothesis if differential thickening occurred before the earliest indication of completed orientation.
MATERIALS AND METHODS
Animals
Animals were handled in accordance with the United States Department of Agriculture Guide for the Care and Use of Animals in Agricultural Research. Pony mares of mixed breeds were used (age, 3–18 yr; weight, 260–410 kg). The mares had free access to grass hay, water, and trace-mineralized salt. Mares were selected that had docile temperament and had no apparent ultrasonically detected abnormalities of the reproductive tract [1]. The selected mares were scanned daily by ultrasound and bred naturally when a preovulatory follicle reached 35 mm and every other day thereafter until ovulation.
Anatomy and Ultrasonography
The transrectal ultrasound transducer was held close to the upper surface of the uterus. In this regard, the equine uterus assumes a T or Y shape as it rests upon other abdominal organs [17]. The stem of the T or Y represents the uterine body, and the arms represent the two uterine horns (Fig. 1). The mesometrium and its accompanying blood vessels attach to the dorsal surface of the horns, as seen in a suspended tract; however, because the uterus usually rests upon other viscera, the attachment to the uterine horns is dorsocaudal with respect to the mare's body. The ultrasound transducer was held approximately parallel to the floor, directed cranially, and rotated on its vertical axis to obtain a cross-sectional view of the vesicle and uterine horn by using a symmetrical image of the horn (Fig. 1). The left aspect of the ultrasound image represented the tissues on the cranial aspect of the transducer. Therefore, the mesometrial attachment was in the upper right corner of the image, even though the anatomical attachment was to the dorsal aspect of the horn. Large branches of blood vessels formed a network along the upper surface of the horns, as seen on the ultrasound image (Fig. 1).
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Pulse-wave ultrasound scanners with both B-mode (gray scale) and color-Doppler functions equipped with 7.5-MHz transducers were used (Aloka SSD-2000 and Aloka SSD-3500; Aloka America, Wallingford, CT). One transducer was a convex array with a beam-field width at the transducer face of 20 mm (UST-995–7.5). The other was a linear array with a beam-field width of 60 mm (UST-5821–7.5). Settings for B-mode and color-Doppler mode controls were kept constant, except for the controls for magnification and delineation of the color-Doppler area. The color-Doppler settings for velocity range and flow filter were 10 cm/sec and 4, respectively. The principles and techniques of Doppler ultrasound in equine reproduction have been reported [18].
End Points
The day of ovulation in these experiments was the day the ovulatory follicle was no longer present at a daily examination and was designated Day 0. Embryo mobility was assessed by assigning vesicle location to one of nine uterine segments (Fig. 1) every 5 min for 2 h, as described [19]. A mobility trial was done each day until the vesicle did not move between uterine segments during 2 h, and the absence of movements was defined as fixation. Fixation was confirmed in all mares on the subsequent days by the continued presence of the vesicle in the same uterine segment. For the study of orientation, sites at the periphery of the cross-sectional image of the embryonic vesicle were assigned positions, according to the face of a clock (Fig. 2), as described [1]. Twelve o'clock was at the center of the mesometrial attachment to the dorsal aspect of the horn. The "o'clock" approach was used for determining the position of the embryo proper at the periphery of the vesicle (Experiments 1 and 2) and for measuring endometrial thickness at various positions (Experiment 2). The position was recorded in an individual to the nearest hour and the average for a group of mares was expressed by hour and tenth of an hour (e.g., 6.5 for 6:30).
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During a preliminary color-Doppler study, it was noted that a colored spot on the ultrasound image, indicating blood flow, developed consistently after fixation in the endometrium at the ventral aspect of the vesicle opposite to the mesometrial attachment and adjacent to the wall of the vesicle. The localized colored area was distinguishable from other endometrial colored spots by closer proximity to the vesicle and greater stability in location and appearance between frequent examinations at a given session (Fig. 3). After completion of Experiment 1, it was clear that these spots indicated the position of the embryonic disc before the embryo proper with heartbeats was identified by B-mode and color-mode scanning. The spots were therefore defined as an early indicator of the future position of the embryo proper. The center of the early indicator was used to record its position relative to the periphery of the vesicle. End points common to both experiments were days of fixation and first detection of the early indicator and the embryo proper, length of intervals between end points, and clock-face positions and day-to-day position changes of the early indicator and embryo proper.
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Experiment 1
Twenty-four mares with embryonic vesicles were used. Embryo mobility trials began on Day 13 and continued daily until the day of fixation. Orientation of the early indicator and the embryo proper were compared among first and last days of detection of the indicator, first day of detection of the embryo proper, and Day 21 (last day of examinations).
Experiment 2
Eleven mares with embryonic vesicles were examined daily from Day 12 until Day 19. A 2-h mobility trial was performed until the vesicle remained in the same uterine segment for 2 h (fixation). Detection of the early indicator was attempted daily during the mobility phase beginning on Day 14.
Endometrial thickness (distance between the myometrium and embryonic vesicle) was measured in B-mode in the caudal segment of the uterine horns daily during the mobility phase and after fixation. During the mobile phase, measurements were made after the vesicle remained for 
5 min in the caudal segment of either horn and were made every 5 min until the vesicle left the segment. Beginning on the day of fixation, measurements were done each day at the site of the embryonic vesicle. Measurements were made from a frozen image of the embryonic vesicle in cross-section relative to the uterine horn at maximum diameter of the vesicle. Thickness measurements of the endometrium were taken at the following clock face positions relative to the site of the mesometrial attachment at 12:00 (center of attachment): 1:30, 4:30, 7:30, and 10:30 (Fig. 2). The clock face positions of 1:30 and 10:30 were defined as representing the dorsal aspect (mesometrial) of the cross-section of endometrium and 4:00 and 7:30 as the ventral aspect (antimesometrial). Thickness was measured with the calipers of the ultrasound scanner between the internal surface of the wall of the embryonic vesicle and the apparent junction between endometrium and myometrium. The internal wall of the vesicle was used because the surface between the endometrium and vesicle wall often was indistinct. The junction between endometrium and myometrium was based on the characteristic echotexture of the endometrium [1].
Dorsal endometrial encroachment upon the vesicle from greater endometrial thickness dorsally than ventrally was assessed by dividing the dorsal thickness measurements by the ventral measurements, and the result was defined as an encroachment ratio (Fig. 2). A ratio of 1 indicated that the dorsal and ventral endometrial walls encompassing the vesicle were similar in thickness, and a ratio of 2 indicated that the dorsal wall was twice as thick as the ventral wall. To reduce the error associated with the irregular shape of the vesicle, the mean of the quotients between 1:30 and 7:30 and between 10:30 and 4:30 was used for each daily value. The encroachment ratio was not different (P > 0.05) among the measurements that were made at 5-min intervals, and the mean for the 5-min intervals was used for each mare and day in further analyses.
Endometrial perfusion or vascularity was evaluated in color-Doppler mode in the caudal segment containing the vesicle during endometrial-thickness measurements. During the mobility phase, the estimates were made when the vesicle was temporarily in the caudal segment of either uterine horn. Perfusion was estimated subjectively by scoring the extent of colored areas in the endometrium during real-time cross-sectioning in a continuous span of 1 min. Because of animal and uterine movements, multiple cross-sections were viewed until an adequate uninterrupted 1-min series was obtained. Only the colored areas that appeared to be within the endometrium were considered. The scores were 1, 2, 3, and 4, indicating nil, minimal, intermediate, and maximal perfusion, respectively. The scoring system was validated previously [8]. The selected 1-min scan was recorded on a digital videocassette.
Vascular perfusion of the endometrium also was assessed objectively by off-line measurement of the number of colored pixels and the number of colored spots as an indicator of blood-flow area, as described [8]. A selected still frame (image) with the estimated maximum flow area from each 1-min recorded scan was used. The images were captured from the videocassettes using Adobe Premiere Pro 1.5 software (TIFF format; Adobe Systems). Colored spots or pixel aggregates were selected from the images, extracted, and saved in GIF format using Adobe PhotoShop 5.5 software (Adobe Systems). The number of colored spots was extracted directly from the GIF format images. ImajeJ 1.31v software (National Institutes of Health) was used for calculation of the total number of colored pixels for each GIF-format image. The subjective and objective perfusion end points were not different (P > 0.05) among the measurements at 5-min intervals, and the mean for the 5-min intervals was used in the analyses for each approach.
Sequential endometrial encroachment and perfusion data were centralized to fixation and extended from 4 days before to 3 days after fixation. Before fixation, daily comparisons of endometrial thickness and vascular perfusion surrounding the vesicle when in a caudal segment were made between the horn of future fixation and the opposite horn. The two horns were compared for 4, 3, and 2 days before fixation; data were not included for the opposite horn on the day before fixation, because of the absence of a vesicle in the opposite horn during the mobility trial in seven of nine mares. Endometrial encroachment was also centralized to first detection of the early indicator.
Statistical Analyses
Data were examined for normality with the Kolmogorov-Smirnov test. Data that were not normally distributed were transformed to natural logarithms. The scores for Doppler vascularity were considered to be nonparametric and were analyzed by a ranking procedure [20]. The ranked scores and the parametric end points were analyzed by the mixed procedure of SAS (version 8.2; SAS Institute, Inc.) to determine the main effects and the interaction, using a repeated statement to account for autocorrelation between sequential measurements. Paired and unpaired Student t-tests were used to locate differences within and between horns, respectively, when significant main effects or an interaction were obtained. Discrete data were analyzed by Student t-tests. The proportion of mares with the embryonic vesicle located in the future horn of fixation versus the opposite horn 1 day before fixation was compared with a chi-square test. A probability of P 
0.05 indicated a significant difference. All values, unless otherwise stated, are the mean ± SEM.
RESULTS
Five mares were not used, because of embryonic loss during the experimental periods (n = 3) and fixation in the uterine body (n = 2), resulting in 21 mares for Experiment 1 and 9 mares for Experiment 2. The mean day of fixation, day of first detection of the early indicator of the future position of the embryo proper, day of first detection of the embryo proper, intervals between these three events, and clock-face position and day-to-day changes in position of the early indicator and embryo proper are shown for each experiment and combined for the two experiments (Table 1). Data from last day of examination in Experiment 2 are not given in the table because of early termination of the experiment (Day 19). The early indicator was first detected on the day of fixation (17/30 mares; 57%) or 1 day (40%) or 2 days (3%) after fixation, resulting in the interval of 0.5 ± 0.1 days between the two events. In all mares in both experiments, the early indicator was detected before detection of the embryo proper (range of intervals, 1 to 5 days). The days of first detection of the embryo proper with heartbeats (number of mares) were as follows: Day 17 (1), Day 18 (11), Day 19 (13), and Day 20 (5).
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Experiment 1
The early indicator served to mark the future position of the embryo proper and was detected in all mares. The early indicator consisted of colored spots in close apposition to or impinging upon the wall of the vesicle (Fig. 3). One to six processes of the colored spots appeared to involve the vesicle wall. The indicator persisted in approximately similar form from first detection until the embryo-proper stage in individual mares. An echoic spot without a color-Doppler signal arose from the internal wall of the vesicle and protruded into the fluid area of the vesicle at the site of the early indicator in 9/21 mares (43%) on the day before an embryonic heartbeat was detected.
The position (clock-face hours) of the early indicator at the first detection did not differ from the position at last detection (day before detection of embryo proper) or from the position of the embryo proper at first detection and at last detection on Day 21 (Table 1). The change in position between first detection of the early indicator and embryo proper did not differ between mares, with an interval from indicator to embryo proper of 1 or 2 days (change in position, 1.1 ± 0.5 h; n = 8) versus 3 days (0.8 ± 0.2 h; n = 13).
Experiment 2
The diameter (cross-sectional relative to the uterine horn) of the embryonic vesicle increased progressively (significant increase each day) from 4 days before fixation (9.8 mm) until the day of fixation (24.1 mm). Growth rate of the vesicle during this time was 3.6 mm/day. Significant vesicle diameter changes were not detected for the 3 days after fixation. During the 2-h mobility trials at 4–2 days before fixation, the percentage of mares in which the vesicle appeared in both horns (39%), only in the future horn of fixation (26%), or only in the opposite horn (35%) was not different among these three groups. However, 1 day before fixation, the vesicle was in only the future horn of fixation or the adjacent cranial end of the uterine body during the mobility trial in 7 of 9 mares, compared to 2 of 9 in only the opposite horn and 0 of 9 in both horns (P < 0.05). An early indicator of the future position of the embryo proper was not identified during the mobility phase in any mare.
The endometrial encroachment ratios, vascularity scores, number of colored spots, and number of colored pixels for 4 days before to 3 days after fixation are shown (Fig. 4). There were no significant effects of horn (horn of future fixation and opposite horn), day, or an interaction of day and horn for the encroachment ratio over 4, 3, and 2 days before fixation. However, for 4, 3, and 2 days before fixation, the main effect of day was significant for vascularity score (P < 0.0001), number of colored spots (P < 0.001), and number of colored pixels (P < 0.005). There was no horn effect. The days of significant differences are shown (Fig. 4). Analyses of data for the horn of fixation from 4 days before to 3 days after fixation were significant (P < 0.003) for each of the four end points. The first significant increase occurred between 4 and 1 days before fixation for the encroachment ratio (P < 0.02), between 4 and 2 days before fixation for vascularity score (P < 0.05) and number of colored pixels (P < 0.05), and between 4 and 1 days before fixation for number of colored spots (P < 0.05).
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The endometrial encroachment ratio in the horn of fixation differed (P < 0.0001) over days centralized to the first day of detection of the early indicator. The ratio first increased (P < 0.04) between 4 and 2 days before first detection of the indicator (Fig. 5).
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DISCUSSION
The diameter growth rate of the embryonic vesicle (3.6 mm/day) during the mobility phase and the day of fixation is similar to reported values [1]. The absence of an increase in cross-sectional expansion of the vesicle relative to the uterine horn after fixation is consistent with previous studies [1]; expansion occurs longitudinally along the uterine lumen because of turgidity of the uterine wall. Fixation occurred in the caudal segment of a uterine horn in 30 of 32 mares. The remaining fixations (6%) occurred in the uterine body, which is similar to the 7% incidence of fixation in the body in a previous study [9]. One of the two mares was bred in two earlier studies, and fixation during each of the three pregnancies occurred in the middle of the uterine body, indicating repeatability.
During the last day of the mobility phase, the vesicle was mobile within the future horn of fixation but did not enter the opposite horn in a significant number of mares (7 of 9). This is a novel finding, and the underlying mechanism is not known. However, the vesicle may have entered the cranialmost aspect of the uterine body before returning to the caudal segment of the horn of fixation; this could not be determined, critically. The slight, but significant, increase in differential dorsal endometrial thickening (encroachment ratio) in the caudal segment of the future horn of fixation between 4 and 1 days before fixation and the reported increased uterine turgidity [9] may have contributed to the fixation process. The degree of dorsal thickening in the nonfixation horn could not be evaluated in the seven mares with apparent confinement of the mobile vesicle in or near the horn of future fixation on the day before fixation; the absence of the vesicle in the caudal segment of the opposite horn precluded measurement of thickening. Further study with a different approach will be needed. Moreover, study is needed on the extent of differential dorsal endometrial thickening in other parts of the horn of fixation and throughout the opposite horn.
In a recent study [8], the extent of vascular perfusion was assessed in the middle segment of each horn during the mobility phase. Perfusion increased in both horns during Days 11–14, but was greater in the horn that contained the embryo at the time of assessment. An increase in vascularity occurred within 7 min after the vesicle entered a horn. In the present study, vascular perfusion during the mobility phase was assessed in the caudal segment of each horn after the vesicle was present in the caudal segment for at least 5 min; the results were similar to those of the previous study [8]. The increase in vascularity began before the increase in the encroachment ratio in the endometrium at the site of future fixation. The postfixation 2-day increase in vascularity score at the site of fixation was also similar for the present and previous studies. However, the increase in the encroachment ratio between 1 and 3 days after fixation in the present study was more rapid than during 4–0 days before fixation, whereas a continued increase in vascularity did not occur 1–3 days after fixation. The nonsynchronous occurrence of endometrial perfusion and encroachment suggested, although tenuously, that the two events were from different stimulating factors, presumably from the embryonic vesicle. The rapid increase in the encroachment ratio after fixation may have reflected the continued presence of the vesicle, assuming that the vesicle produced a factor that stimulated thickening of the endometrium. It seems reasonable that the differential thickening would occur closest to the mesometrial attachment or the point of entry of blood vessels into the uterine horns. In this regard, the dorsal thickening may be considered preparatory to the attachment of the umbilical cord adjacent to the mesometrial attachment during the fetal stage [14].
Morphology of the fixation site (implantation chamber) has been examined with histologically-prepared specimens [21]. As the vesicle expanded, the ventral endometrium at the embryonic pole dilated and formed the chamber. The endometrial folds were distinct dorsally and flattened ventrally. The chamber assumed an oval shape, indicating that the turgid horn allowed expansion only in the direction of the uterine lumen. Endometrial folds at each end of the chamber were closely apposed. These descriptions from excised tracts are consistent with earlier descriptions based on ultrasonography [12]. However, morphologic description of the tissue architecture of the profound hypertrophy at the dorsal aspect of the chamber apparently has not been reported. The echotexture of the endometrial encroachment was not considered or compared to the echotexture of the endometrium in other parts of the uterus in the present study. Comparison of the dorsal and ventral aspects of the endometrium by ultrasound in the present study was not productive, primarily because enhancement artifacts obscured the endometrium beneath the fluid-filled vesicle; enhancement artifacts result from relative oversaturation by echoes from the ventral wall because of limited attenuation of the ultrasound beams while passing through the yolk-sac fluid [22]. The morphology and underlying mechanisms involving the endometrial enlargement near the mesometrial attachment will need investigation.
The discovery of an early color-Doppler indicator in the endometrium marking the future position of the embryo proper at the periphery of the vesicle in all 30 mares was an asset in the study of the orientation phenomenon. Results supported the hypothesis that dorsal endometrial encroachment begins before the earliest indication that orientation had occurred and, in addition, indicated that encroachment began even before fixation.
The range of intervals from first detection of the early indicator to the first detection of the embryo proper (1 to 5 days) indicated the extent of variation in the interval. Nevertheless, the mean clock-face position of the early indicator and the embryo proper remained approximately constant and were not different from one another. The day-to-day change in position of the early indicator and embryo proper in individuals throughout the experiments was minimal (means of 0.4 and 0.2 clock-face hours). That is, there was little position orientation change from day-to-day or between first detection of the early indicator and the last examination of the embryo.
The anatomical origin of the color-Doppler signals that were used as early indicators of the future position of the embryo proper, and especially the colored processes that appeared to permeate the vesicle wall, are unknown. The major portion of the colored area likely represented blood flow in endometrial vessels that were stimulated or dilated as a result of the close apposition to the embryonic disc. The color processes that appeared to reach the yolk-sac fluid may have represented movement of blood in rudimentary embryonic vessels and contractions of the primitive heart. In this regard, a detailed study of embryonic angiogenesis at this stage is lacking. It has been reported that an anastomotic network of blood vessels in the yolk-sac wall adjacent to the embryo proper and a heart chamber were present on Day 18, but the crucial Days 15–17 were not included in the study [23]. The color responses at the edge of the colored spots also may have represented blooming artifacts or the extension of the color signals beyond the lumens of blood vessels [24]. Our current technology and knowledge were not adequate for determining whether portions of the colored early indicator involved embryonic as well as endometrial vessels, and further study is needed.
First detection of the embryo proper with heartbeats (on Days 17–20) was about 2 days earlier than for previous reports (Days 19–22 [1]). The earlier detection represented an improvement in the resolution of the B-mode ultrasound technology combined with confirmation of heartbeats with the color-Doppler function. The detection of a small echoic spot on the internal surface of the vesicle at the position of the early indicator in 43% of mares on the day before detection of the embryo proper can be attributed to an embryo proper before the detection of heartbeats.
In conclusion, the embryonic vesicle entered and was mobile in the uterine horn of future fixation on the day before fixation more frequently than for the opposite horn. Differential dorsal thickening of the endometrium that surrounds the embryonic vesicle began during the later days of the mobility phase. After fixation, the differential dorsal thickening or endometrial encroachment upon the vesicle increased rapidly and was more than four times thicker than ventrally by 3 days after fixation. Color-Doppler-imaged blood vessels formed in the endometrium close to the ventral wall of the embryonic pole of the vesicle an average of 0.5 days after fixation and 2.5 days before detection of the embryo proper with heartbeats. These distinct color spots were designated as early indicators of the future position of the embryo proper at the periphery of the vesicle. An early indicator was detected in all mares opposite to the dorsal endometrial thickening and mesometrial attachment, and its position did not differ significantly from the position of the later-detected embryo proper with heartbeats. Based on the position of the early indicator, orientation of the embryonic vesicle occurred immediately after fixation, which on a temporal basis supports the hypothesis that dorsal endometrial encroachment begins before the earliest indication that orientation has occurred.
ACKNOWLEDGMENTS
The authors thank E.L. and M.O. Gastal and M.D. Utt for technical advice and suggestions and B.L. Rodrigues for assistance with animal monitoring and handling.
FOOTNOTES
1 Supported by Eutheria Foundation (Cross Plains, WI); projects P1-LS-03, P2-LS-03, and P1-LS-05. 
2 Correspondence: O.J. Ginther, Animal Health and Biomedical Sciences, 1656 Linden Dr., University of Wisconsin, Madison WI 53706. FAX: 608 262 7420; ginther{at}svm.vetmed.wisc.edu
Received: 19 September 2005.
First decision: 10 October 2005.
Accepted: 17 October 2005.
REFERENCES
This article has been cited by other articles:
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  J C Ferreira, E L Gastal, and O J Ginther Uterine blood flow and perfusion in mares with uterine cysts: effect of the size of the cystic area and age Reproduction, April 1, 2008; 135(4): 541 - 550. [Abstract] [Full Text] [PDF]
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