Effects of the embryo on uterine morphology and function in mares

AnimalReproduction Science, 31 (1993) 311-329

311

Elsevier Science Publishers B.V., A m s t e r d a m

Effects of the embryo on uterine morphology and function in mares P.G. Griffin, E.M. Carnevale and O.J. Ginther Department of Veterinary Science, Universityof Wisconsin-Madison, 1655 Linden Drive, Madison, W153706, USA (Accepted 11 June 1992)

ABSTRACT Griffin, P.G., Carnevale, E.M. and Ginther, O.J., 1993. Effects of the embryo on uterine morphology and function in mares. Anita. Reprod. Sci., 31:311-329. Surgical ligations of the uterine horns on Day 11 (ovulation designated Day 0) were used to restrict the embryonic vesicle to the uterine body and caudal uterine horns; uterine characteristics were assessed in the isolated cornual segments. Transrectal palpation was used to assess uterine tone and ultrasonic scanning was used to assess uterine edema (scores 1-4, minimal to maximal for both end points). Tone was greater (P < 0.05 ) or tended to be greater (P < 0.07 ) for uterine-intact mares (n = 5 ) than for uterine-ligated mares (n = 5) on each of Days 13-19. A significant interaction indicated higher endometrial echotexture scores over Days 17-24 in the uterine-intact mares than in the uterine-ligated mares. In another experiment, cornual ligations were done before Day 11 to restrict the embryo to the side of ovulation (n = 10 ). Significant horn by day interactions indicated that the uterine horn containing the embryo (ipsilateral horn) had greater tone and smaller horn diameter than the contralateral horn over Days 13-23; tone and diameter of the ipsilateral horn were similar to those of pregnant uterineintact mares (n = 6 ). A significant horn by day interaction indicated higher endometrial echotexture scores for the ipsilateral horns in mares with ligations and for both horns in mares without ligations than for the contralateral horn over Days 17-25. Uterine contractility, assessed ultrasonically at the uterine body, was decreased ( P < 0.05 ) in uterine-ligated mares (no conceptus exposure in the uterine body) on each of Days 11-14, relative to uterine-intact mares. Results indicated that the early conceptus promotes an increase in uterine tone with a corresponding decrease in diameter, enhanced endometrial folding, and increased uterine contractility through a local mechanism. That is, a greater effect was detected in the uterine segments exposed to the conceptus or its secretory products than in the segments without conceptus exposure.

INTRODUCTION E a r l y p r e g n a n c y i n m a r e s is m a r k e d b y a c o n t i n u u m of physical interactions between the conceptus and uterus involving: ( 1 ) intrauterine mobility

Correspondence to: O.J. Ginther, Department of Veterinary Science, University of WisconsinMadison, 1655 Linden Drive, Madison, WI 53706, USA.

© 1993 Elsevier Science Publishers B.V. All rights reserved 0378-4320/93/$06.00

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of the early embryonic vesicle (occurring by Day 9; ovulation designated Day 0); (2) fixation of the embryonic vesicle (cessation of embryo mobility on Day 15 or 16); (3) orientation of the vesicle (post-fixation rotation) (Ginther, 1983a,b). In temporal association with the occurrence of these phenomena, a number of profound functional and morphologic changes occur in the uterus. Uterine contractility reaches maximal levels on Days 11-14 (Cross and Ginther, 1988 ) in association with the period of maximal embryo mobility (Leith and Ginther, 1984), uterine tone increases markedly above mid-diestrous levels between Days 12 and 22 (Van Niekerk, 1965; Hayes and Ginther, 1986), and at the same time uterine diameter decreases (Griffin and Ginther, 1991 a). Additionally, an irregular pattern of ultrasonic echotexture, attributable to increased endometrial edema, is sometimes apparent after Day 14 or 15 (Ginther, 1986; Griffin and Ginther, 1991a). It is postulated that the conceptus, through its secretory products, plays an active role in stimulating or enhancing these uterine changes (Ginther, 1986). It is not known whether the effects of the conceptus on the uterus involve direct contact of the mobile embryonic vesicle with the endometrium, distribution of conceptus products throughout the uterus, or systemic pathways. It has been postulated (Ginther, 1983a) that intrauterine mobility of the equine embryonic vesicle has a role in metabolic or physiologic exchange between the vesicle and uterine luminal fluids or endometrium. One such exchange involves the mechanism for prevention of uterine induced luteolysis by the embryo (Ginther, 1985; McDowell et al., 1985 ). It appears that a majority of the endometrium must be exposed to the conceptus for prevention of luteolysis and pregnancy maintenance (McDowell et al., 1985 ). The present experiments were designed to investigate the local versus systemic effects of the early embryonic vesicle on tone, diameter, ultrasonic echotexture, and contractility of the uterus and to study the effects of restriction of embryo mobility on growth, fixation, and orientation of the early conceptus. Specifically, the hypothesis was tested that the early embryonic vesicle locally increases uterine tone and contractility, decreases uterine diameter, and enhances endometrial edema in those uterine segments with which the vesicle or its secretions have direct contact. In a preliminary study (Experiment 1 ), the vesicle was restricted to the caudal uterine horns and uterine body by surgical ligation of the horns on Day 11. In Experiment 2, the uterine horns were surgically ligated on specific days post-ovulation to confirm the findings of Experiment 1, to determine the time of entry of the embryonic vesicle into the uterine body from the uterine horn ipsilateral to the side of ovulation, and, secondarily, to examine the effects of restricted embryo mobility on maintenance of the corpus luteum. Transrectal ultrasonography was used in both experiments to monitor uterine diameter and echotexture, conceptus characteristics, and luteal size. Transrectal palpation was used to evaluate uterine tone.

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MATERIALSAND METHODS

Experiment 1 Twelve pregnant pony mares, 150-300 kg and 3-12 years of age, were used. On Day 11, the mares were randomly assigned to one of two groups (uterineligated, n = 7; uterine-intact, n = 5 ), after ultrasonic detection of an embryonic vesicle. Uterine ligation was done through a ventral midline laparotomy on Day 11. General anesthesia was induced with xylazine ( 1.0 mg k g - 1, i.v. ) followed by ketamine (2.0 mg k g - 1, i.v. ). Mares were placed in dorsal recumbency, and anesthesia was maintained with halothane. Two adjacent, encircling ligatures (No. 3 Braunamid; B. Braun Melsungen AG, W-3508 Melsungen, Germany) were placed tightly around each uterine horn approximately two-thirds of the distance from the utero-tubaljunction to the corpus-comual junction. The ligatures were passed through the broad ligament, with care taken to minimize disturbance of uterine blood supply. Ligatures were placed without knowledge of the intrauterine location of the embryonic vesicle. Beginning on Day 11, each mare was administered 100 mg of progesterone in safflower oil, intramuscularly, daily until Day 25 to replace endogenous progesterone that could be lost owing to luteolysis (McDowell et al., 1985 ). Daily examinations, consisting of transrectal ultrasound scanning and transrectal uterine palpation, were performed on all mares from Day 11 (prior to surgery) to Day 25. Ultrasonic scanning was done with a real-time, B-mode scanner (Tokyo Keiki, LS 300; Products Group International, Boulder, CO) equipped with a 5.0 MHz linear array transducer. Scores and measurements for each end point were determined at the time of examination without knowledge or consideration of values obtained for previous days.

Experiment 2 Thirty-one pony mares, 150-350 kg and 2-15 years of age, were used. Mares were randomly assigned to one of seven groups as follows: ( 1 ) uterine-ligation on Day 4 or 5 ( n = 5 ) , D a y 6 ( n = 4 ) , D a y 7 ( n = 4 ) , D a y 8 ( n = 3 ) , D a y 9 ( n = 4 ), and Day 10 ( n = 4 ); (2) uterine-intact ( n = 7 ). It was expected that the ligations on Days 4 and 5 would precede passage of the conceptus from oviduct to uterus (Freeman et al., 1991 ). All mares were artificially inseminated with raw semen beginning on the day of ultrasonic detection of a follicle at least 30 m m in diameter and continuing every other day until, but not including, the day of ovulation. Uterine ligations were done as for Experim e n t 1, except that ligatures were placed nearer to the uterine body, using the external junction at the medial aspects of the horns as a landmark. Ultrasonic pregnancy diagnoses were begun on Day 11 (Ginther, 1986).

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Mares in which an embryonic vesicle was not detected on Day 11 were reexamined for pregnancy on Days 12 and 15. Non-pregnant mares were not used. Pregnant mares within each treatment group (uterine-ligated, uterineintact) were alternately assigned to one of two groups (progesterone-treated and non-treated) when they reached Day 12. For progesterone-treated mares, daily treatment, as described for Experiment 1, began on Day 12 and continued until Day 25. Daily examinations, consisting of transrectal ultrasound scanning and transrectal uterine palpation, as in Experiment 1, were performed from Day 10 to Day 25. Ultrasound examinations were recorded using a 3 / 4 in. videotape recorder connected to the ultrasound scanner. Uterine tone scores and diameter measurements were determined at the time of examination, as in Experiment 1. Endometrial echotexture and uterine contractility scoring were done from the videotapes subsequent to the examinations. A high-resolution video m o n i t o r was used for tape analysis, and scores for both end points were assigned without knowledge of mare, group assignment, or n u m b e r of days from ovulation.

End points (both experiments) The tone of each uterine horn was assessed digitally and scored on a scale of 1-4 (minimal or flaccid to maximal or turgid), as described (Hayes and Ginther, 1986). In the uterine-ligated groups, the tone of each horn was assessed at the approximate m i d p o i n t between the ligatures and the tip of the uterine horn; in the uterine-intact groups, tone was assessed at a corresponding cornual location. The outer cross-sectional diameter ( m m ) of each uterine horn, defined as the m e a n of maximal height and width, was determined using the electronic calipers of the ultrasound scanner at approximately the same location as for tone assessment. Assessment of uterine tone by transrectal palpation produces results compatible with expected morphological and biological events (review: Ginther, 1986). Even small transient increases in tone have been demonstrated without operator knowledge of reproductive status or day of the estrous cycle or pregnancy (Griffin et al., 1992 ). Furthermore, palpable changes in tone are accompanied by changes in ultrasonically obtained diameters of the uterine horns; the two end points are negatively correlated (Griffin and Ginther, 199 l b). The ultrasonic echotexture of the e n d o m e t r i u m of each uterine horn and the uterine body was assessed and scored on a scale of 1-4, based on the degree of endometrial edema (minimal to maximal), as described (Ginther and Pierson, 1984). This procedure is also reliable even when done from videotapes without knowledge of mare identity; it is used extensively in this laboratory (reviews: Ginther, 1986, 1992 ). Echotexture scores parallel sexual behavior scores (Hayes et al., 1985 ). Ultrasonically detectable uterine contractility was assessed from 1-min scans of longitudinal sections of the uterine body, as described (Cross and Ginther,

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1988). Contractility was scored on a scale of 1-5 (minimal to maximal), based on the extent of to and fro or flowing movements of the endometrium. This procedure produces biological meaningful results that are repeatable with different operators even when done from videotapes without knowledge of mare identity (Cross and Ginther, 1988; Griffin and Ginther, 1990 ). The intrauterine location of the embryonic vesicle was determined at each ultrasonic examination and assigned to one of nine segments (cranial, middie, and caudal thirds of each uterine horn and the uterine body; Ginther, 1984). Fixation of the embryonic vesicle was defined as the first day that the vesicle was in the same uterine segment during every daily examination. Diameter ( m m ) of the vesicle was defined as the mean of maximal height and width. When the embryo proper was first identified, its orientation was assigned a score based on its location relative to the face of an imaginary clock (Kastelic et al., 1987) as follows: Score 1, 6 o'clock; Score 2, 5 or 7 o'clock; Score 3, 4 or 8 o'clock; Score 4, 3 or 9 o'clock; Score 5, 2 or 10 o'clock; Score 6, 1 or 11 o'clock; Score 7, 12 o'clock. The presence and diameter (mean of maximal height and width in millimeters) of the corpus luteum were determined.

Statistical analyses Sequential data for both experiments were analyzed by split-plot analyses of variance using the Statistical Analysis System (SAS) General Linear Models Procedure (SAS, 1985 ). When significant ( P < 0.05 ) effects of day were detected, differences among days were further examined using least significant difference. Differences between groups within a given day were analyzed using t-tests and differences between horns (e.g. ipsilateral vs. contralateral to embryonic vesicle) within a given group and day were analyzed using paired t-tests. For single point data, differences between groups were analyzed using t-tests. Frequency data were examined by Z 2 analyses of Fisher's Exact Test (SAS, 1985 ). The correlation between uterine horn diameter and uterine tone was determined using Pearson's correlation procedure (SAS, 1985 ). RESULTS

Experiment I Upon ultrasonic examination on Day 12, the embryonic vesicle in five mares was trapped caudal to the ligatures (i.e. vesicle restricted to the uterine body and caudal uterine horns). In one mare, the vesicle was trapped cranial to the ligatures in the left uterine horn (contralateral to the side of ovulation) and, in another mare, the vesicle may have been crushed during surgery as it was undetected thereafter; neither mare was considered in the analyses. The re-

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suiting number of mares was five for the uterine-ligated group (no contact of embryonic vesicle with the cranial and middle segments of either uterine horn), and five for the uterine-intact group (no restriction of vesicle mobility ). On the last day of the study (Day 2 5 ), ultrasonically detectable constriCtions were present in the horns of all uterine-ligated mares in the expected areas of the ligatures. For both groups, there were no differences between right and left uterine horns for uterine tone score, horn diameter, or echotexture score; therefore, values averaged over both horns were analyzed and are shown in all figures. Mean uterine tone scores and mean uterine horn diameters are shown in Fig. 1, along with results of the statistical analyses. Within the uterine-intact group, 4 G: P<0.1 D: P<0.0001 GD: P
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Fig. 1. Mean ( _+SEM) uterine tone scores and uterine horn diameters for uterine-intact and uterine-ligated mares (Experiment 1 ). In uterine-ligated mares, the conceptus was prevented from entering the portions of the uterus that were assessed for tone and diameter. Tone was assessed digitally and scored on a scale of 1-4 (minimal or flaccid to maximal or turgid). Within each group, tone score and horn diameter were negatively correlated ( P < 0.0001; uterine-intact, r = - 0 . 6 0 ; uterine-ligated, r = - 0 . 6 4 ) . G, group effect; D, day effect; GD, group X day interaction; NS, not significant.

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there were increases ( P < 0.05 ) in tone scores between Days 11 and 13, 13 and 17, and 18 and 25 and decreases ( P < 0 . 0 5 ) in horn diameter between D a y s 11 and 14, and 13 and 22. Within the uterine-ligated group, there were increases ( P < 0.05 ) in tone scores b e t w e e n D a y s 16 and 19 and 19 and 23, b u t there were no significant changes before D a y 16; a significant effect o f day was not detected for uterine horn diameter. Uterine tone scores were greater ( P < 0.05 ) or t e n d e d to be greater ( P < 0.07) for uterine-intact mares than for uterine-ligated mares on each o f D a y s 13-25, except on Days 20 and 23. Uterine horn diameters t e n d e d to be greater ( P < 0.1 ) for uterine-ligated mares than for uterine-intact mares on Days 15 and 22. M e a n endometrial echotexture scores and results o f statistical analyses are shown in Fig. 2. M e a n echotexture scores averaged over b o t h uterine horns were greater ( P < 0.05) for the uterine-intact group than for the uterine-ligated group on each o f D a y s 17-24. There was no difference between groups in echotexture scores for the uterine body. There was no effect o f group or a group × day interaction b e t w e e n D a y s 11 and 25 for uterine contractility scores, luteal diameter, or e m b r y o n i c vesicle diameter. F o u r o f five mares in the uterine-ligated group and five o f five mares in the uterine-intact group h a d an ultrasonically detectable corpus luteum on D a y 25 ( m e a n + S E M diameter, 17.9__+0.9 m m vs. 1 8 . 4 + 0 . 9 m m , respectively; no significant difference). The m e a n + SEM day o f fixation for uterine-ligated and uterine-intact mares was 13.8 + 0.6 days and 14.6 + 0.9 days, respectively (no significant difference). In all mares in b o t h groups, the ve-

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Fig. 2. Mean ( _+SEM) endometrial echotexture scores averaged over both uterine horns for uterine-intact and uterine-ligated mares (Experiment 1). In uterine-ligated mares, the conceptus was prevented from entering the portions of the uterine horns that were assessed for echotexture. Scoring was done on a scale of 1-4, based on the degree ofendometrial edema (minimal to maximal). G, group effect; D, day effect; GD, group × day interaction.

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side became fixed in the caudal segment of one of the uterine horns. There was no difference in mean orientation scores between uterine-ligated and uterine-intact mares (2.6 _ 1.1 vs. 2.6 _ 0.5, respectively). For all mares in both groups, embryonic heartbeats were detected and embryonic vesicles seemed to have a normal ultrasonic appearance on Day 25 (Ginther, 1986 ).

Experiment 2 Pregnancy was detected in 11 uterine-ligated mares and six uterine-intact mares. In all but one uterine-ligated mare, the embryonic vesicle was trapped cranial to the ligatures in the uterine horn ipsilateral to the corpus luteum. In the exception (ligation on Day 10), the vesicle was restricted to the uterine body; data for this mare were excluded from all analyses of uterine end points. TABLE1 Pregnancy rates, conceptus loss rates, and conceptus characteristics (mean _+SEM ) in uterine-ligated and uterine-intact mares (Experiment 2) End point

Pregnancy rate at Day 12 Day of ligation 1 4 or 5 6 7 8 9 10 All mares 2 Day of fixation 3 Orientation score4 Embryo diameter (ram) on day o f fixation 3 Embryo loss rate by Day 255 Day of embryo loss

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3/5 (60%) 4/4 (100%) 1/4 (25%) 0/3 (0%) 1/4 (25%) 2/4 (50%) 11/24 (46%) 13.3 + 0.3 2.7 + 0.5

6/7 (86%) 16.0 + 0.4 1.8 + 0.3

11.5 _+2.0 6/11 (55%) 18.5 +_ 1.2 (range 15-22)

22.4 + 1.9 2/6 (33%) 16.5 + 2.5 (range 14-19)

'Overall, there was no significant effect of day of ligation on pregnancy rates. In all but one mare (ligation on Day 10), the embryonic vesicle was trapped cranial to the ligatures in the uterine horn ipsilateral to the corpus luteum. 2Rates tended ( P < 0.06) to be different between groups. 3Significant difference between groups for a given end point. 4Orientation of the embryo proper on the ultrasound image was scored as follows: Score 1, 6 o'clock; Score 2, 5 or 7 o'clock; Score 3, 4 or 8 o'clock; Score 4, 3 or 9 o'clock; Score 5, 2 or 10 o'clock; Score 6, 1 or 11 o'clock; Score 7; 12 o'clock. 5In three of six uterine-ligated mares and two of two uterine-intact mares in which embryo loss occurred, the corpus luteum was ultrasonically detectable on Day 25. Two of six losses in uterine-ligated mares and both losses in uterine-intact mares occurred in progesterone-treated mares.

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Fig. 3. Mean ( _+SEM) uterine tone scores and uterine horn diameters for uterine-ligated and uterine-intact mares (Experiment 2). In uterine-ligated mares, the embryonic vesicle was confined to the ipsilateral uterine horn. Tone was assessed digitally and scored on a scale of 1-4 (minimal or flaccid to maximal or turgid). Tone score and horn diameter were negatively correlated ( P < 0.0001 ) between Days 10 and 25 (uterine-ligated, ipsilateral horn, r = - 0.55; contralateral horn, r = - 0 . 4 5 ; uterine-intact, r = - 0 . 6 3 ) . G, group effect; D, day effect; GD, group × day interaction; NS, not significant.

For all uterine-ligated mares, an ultrasonically detectable constriction was present at the expected site of ligation on each day of the study. Pregnancy rates, conceptus loss rates, and conceptus characteristics are shown in Table 1. The low pregnancy rate ( 11/24, 46%) and high embryo loss rate (6/11, 55%) for uterine-ligated mares and the embryo loss rate in uterine-intact mares (2/6, 33%) prevented statistical handling of data on effects of progesterone treatment; however, inspection of the data indicated no apparent effects and data for progesterone-treated and non-treated mares in both groups were combined. The resulting n u m b e r of pregnant mares was 10 for the uterineligated group (conceptus restricted to one uterine h o r n ) , and six for the uterine-intact group (no restriction of embryo mobility). For uterine-ligated and

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Fig. 4. Mean ( + SEM) endometrial echotexture scores for uterine-ligated and uterine-intact mares (Experiment 2). Echotexture scoring was done blind on a scale of 1-4 (minimal to maximal endometrial edema). In uterine-ligated mares, the embryonic vesicle was confined to the ipsilateral uterine hom. G, effect of group; D, effect of day; GD, group × day interaction; NS, not significant. uterine-intact mares in which embryo loss occurred, data for uterine end points were considered only for days in which an embryonic vesicle was detected in the presence o f an ultrasonically detectable corpus luteum; data for days subsequent to embryo loss or when a corpus l u t e u m was not detected were not considered for analysis. For uterine-intact mares, but not for uterine-ligated mares, there were no effects or interactions for any end point involving horn side (same or opposite to the side o f ovulation or side o f vesicle fixation) or uterine segment (uterine horns vs. uterine b o d y ) . Therefore, for uterine-intact mares, values for each end point were averaged over both horns or both horns a n d the uterine b o d y for statistical analyses. For analyses involving both uterine-ligated

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and uterine-intact mares, each uterine horn and the uterine body of uterineligated mares were treated independently. Mean uterine tone scores and uterine horn diameters for Days 10-25 are shown in Fig. 3, along with results of statistical analyses. For uterine-ligated mares, uterine tone scores for the horn containing the embryonic vesicle (ipsilateral h o r n ) i n c r e a s e d ( P < 0.05 ) between Days 11 and 12, 12 and 15, 15 and 18, and 18 and 23. Tone scores for the horn not containing the vesicle (contralateral horn) increased ( P < 0.05 ) between Days 17 and 22 and 22 and 24; no significant changes occurred prior to Day 17. Diameters for the ipsilateral horn decreased ( P < 0.05 ) between Days 13 and 17 and 17 and 20. Diameters for the contralateral horn decreased ( P < 0.05) between Days 16 and 18 and Days 18 and 24, with no significant changes prior to Day 16. Tone scores were greater ( P < 0.05 ) for the ipsilateral horn than for the contralateral horn on each of Days 12-23; diameters for the ipsilateral horn were smaller ( P < 0.05) or tended ( P < 0.1 ) to be smaller than for the contralateral horn on each of Days 13-23, except on Day 17. For uterine-intact mares, tone scores averaged over both horns increased ( P < 0.05) between Days 10 and 12, 12 and 14, 14 and 16, and 16 and 19; diameters decreased ( P < 0 . 0 5 ) between Days 10 and 12, 12 and 16, 16 and 22, and 22 and 24. Tone scores and diameters for the ipsilateral horn did not differ from scores or diameters

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averaged over both horns for uterine-intact mares. Tone scores were greater ( P < 0.05) or tended ( P < 0.1 ) to be greater for uterine-intact mares than for the contralateral horn of uterine-ligated mares on each of Days 13-25; diameter of the contralateral horn was smaller ( P < 0.05 ) or tended ( P < 0.1 ) to be smaller than for uterine-intact mares on each of Days 13-16. Mean endometrial eehotexture scores for Days 10-25 are shown in Fig. 4, along with results of the statistical analyses. For uterine-ligated mares, echotexture scores were greater ( P < 0.05 ) or tended ( P < 0.1 ) to be greater for the ipsilateral horn than for the contralateral horn on each of Days 11-25, except on Days 16 and 20. There were no differences in echotexture scores for the ipsilateral horn of uterine-ligated mares and scores averaged over both horns for uterine-intact mares. Echotexture scores averaged over both horns for uterine-intactmares were greater ( P < 0.05) than for the contralateral horn of uterine-ligated mares on each of Days 17, 20-22, 24, and 25; scores tended ( P < 0.1 ) to be greater on Days 11 and 18. Scores for the uterine body were greater ( P < 0.05 ) for uterine-intact mares than for uterine-ligated mares on Days 19 and 21 and tended ( P < 0.1 ) t o be greater on Day 18. Mean uterine contractility scores are shown in Fig. 5, along with results of the statistical analyses. Contractility scores were greater ( P < 0.05 ) for uterine-intact mares than for uterine-ligated mares on each of Days 11-14. There was no effect or interaction involving group (uterine-ligated vs. uter-

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ine-intact) on diameter of the embryonic vesicle between Days 10 and 25. In all uterine-intact mares, the vesicle became fixed in the caudal segment of one uterine horn. In all but one uterine-ligated mare, the vesicle became fixed in the caudal segment of the ipsilateral horn, immediately adjacent to the ligatures; in the remaining mare, the vesicle became fixed at the tip of the uterine horn. Mean luteal diameters are shown in Fig. 6, along with results of statistical analyses. The corpus luteum was ultrasonically detectable on Day 25 in eight of 10 uterine-ligated mares and six of six uterine-intact mares diagnosed as pregnant on Day 12. Luteal diameters for uterine-ligated mares were smaller ( P < 0.05) or tended ( P < 0.1 ) to be smaller than for uterine-intact mares on each of Days 15-25, except on Days 16 and 22. For uterine-ligated mares, but not for uterine-intact mares, there was a significant effect of day on luteal diameter. For uterine-ligated mares, luteal diameter decreased ( P < 0.05 ) between Days 10 and 16 and Days 16 and 18. DISCUSSION

Conceptus The equine embryo first enters the uterine horn from the oviduct between 130 and 142 h post-ovulation (i.e. on Day 6; Freeman et al., 1991 ). In some mares with a large embryonic vesicle, first ultrasonic detectability (vesicle diameter, 2 or 3 m m ) occurs on Day 9 or 10 when the vesicle is usually found (60% of the time) in the uterine body (Leith and Ginther, 1984 ). The vesicle first enters the uterine body, therefore, sometime between Days 6 and 10. In Experiment 2, the pregnancy rate for mares with uterine ligation on Days 710 (4/15, 27%) was lower ( P < 0 . 0 3 ) than for mares with ligation on Days 4-6 (7/9, 78%). The small number of diagnosed pregnancies for mares with ligation after Day 6 precluded critical determination of the day of embryo entry into the uterine body. However, in the one mare diagnosed pregnant after ligation on Day 7, the embryonic vesicle was in the uterine horn ipsilateral to the corpus luteum. Perhaps the low pregnancy rate for mares with ligation on Days 7-9 (2/11, 18%) was due to an intrauterine location of the vesicles in the vicinity of ligatures at the time of surgery, resulting in potential exposure to trauma. If this assumption is correct, first entry of the vesicle into the uterine body probably occurs after Day 8 or, coincidentally, about the time it first becomes detectable by ultrasonic imaging. Diameter of embryonic vesicle on Days 11-25 was not different between uterine-ligated and uterine-intact mares in either experiment. This result suggests that the ligation procedure did not retard embryonic development either by limiting embryo mobility or by secondary effects (e.g. interference with uterine circulation, release of toxins). The conceptus had apparently enough

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exposure to the endometrium to meet its early metabolic needs. Similarly, in an earlier study (McDowell et al., 1988 ), restriction of embryo mobility apparently did not affect vesicle size, although this was not examined critically. The day of fixation of the embryonic vesicle was not affected by uterine ligation in Experiment 1, but was hastened in Experiment 2. In both experiments, the expected site of fixation in uterine-intact mares (i.e. a caudal cornual segment; Ginther, 1983b) was available to the conceptus in uterine-ligated mares. The reasons for the differences between experiments are not known. In Experiment 1, the portion of the uterus available to the mobile embryo (i.e. uterine body and caudal horns) may have been larger in diameter than the available uterine segments in Experiment 2 (i.e. cranial, middle, and caudal segments of the ipsilateral horn ). In a previous study (Griffin and Ginther, 1991b), uterine diameter increased progressively from cranial to caudal cornual segments in early pregnant, uterine-intact mares. In addition, in Experiment 2, the ligations at the caudal aspect of the uterine horn may have resulted in decreased horn diameter for some distance cranial to the site of ligation. The earlier fixation in Experiment 2, therefore, may have been a result of smaller uterine diameter. It has been postulated (Ginther, 1985) that the timing of fixation is a function of increasing uterine tone, decreasing uterine diameter, and expansion of the embryonic vesicle. Uterine tone and horn diameter

The present studies indicated that restriction of the conceptus to physically isolated uterine segments resulted in maintenance of diestrous levels of uterine tone and diameter in the uterine segments without conceptus exposure at a time when tone was increasing and diameter decreasing in segments that contained the conceptus or in the uterine horns of uterine-intact mares. The early conceptus, therefore, affected uterine tone and diameter through a local mechanism (i.e. effects occurred only in segments with conceptus exposure), supporting the stated hypothesis. The changes in tone and diameter observed for uterine-intact mares and for the ipsilateral horn of uterine-ligated mares (Experiment 2) are consistent with those previously reported for early pregnant mares (Van Niekerk, 1965; Hayes and Ginther, 1986; Griffin and Ginther, 1991a). The present findings indicate that physical contact between the conceptus, or conceptus products, and the endometrium is necessary for the characteristic changes in uterine tone and diameter observed during early pregnancy. The nature of this stimulus is not known. However, the early equine conceptus is capable of synthesizing and secreting estrogens and other steroids (Zavy et al., 1979; Marsan et al., 1987), prostaglandins (Watson and Sertich, 1989), and numerous peptides and uncharacterized factors (Fazleabas and McDoweU, 1983; Sharp et al., 1989), the roles of which await clarification. In

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steroid-treated mares, the turgid uterine tone of early pregnancy was most closely mimicked b y a daily regimen of estrogen and progesterone, subsequent to a period of progesterone priming (Hayes and Ginther, 1986). Similarly, the daily administration of estrogen to early pregnant mares hastened the day of fixation, when compared with control mares (Bessent et al., 1988 ). There is a close temporal relationship between the development of turgid uterine tone and the day of fixation of the embryonic vesicle (Ginther, 1985 ). Perhaps, as has been postulated (Ginther, 1986), estrogens of conceptus origin are responsible, through a local mechanism, for the profound increases in uterine tone and decreases in uterine diameter which, along with expansion of the conceptus, result in vesicle fixation. Interestingly, after Day 16, tone and diameter of the uterine segments without conceptus exposure began to increase and decrease, respectively, in parallel with those observed for segments exposed to the conceptus (Figs. 1 and 3). These observations suggest that after Day 16, the local effects of the conceptus are supplemented or replaced by a systemic mechanism unrelated to the location of the embryonic vesicle. The source of the stimulus for systemic control of tone m a y be the conceptus or structures not of conceptus origin (e.g. ovarian follicles or corpus luteum). This apparent shift from a local to systemic mechanism in the control of uterine tone and diameter has not been described previously and requires confirmation. Temporally, the observed shift from a local to a systemic mechanism was associated with the expected time of fixation of the embryonic vesicle in uterine-intact mares (Ginther, 1983a). This observation is consistent with the assumption that the local control of uterine tone by the conceptus involved the embryo-mobility phenomenon, ARernatively, the tone and diameter changes which occurred after Day 16. in the uterine segments without conceptus exposure could have been due to the development of leakage of conceptus products through the ligatures. This seems unlikely, however, because echotexture changes during this time remained confined to the vesicle-containing segments (Figs. 2 and 4 ) . For uterine-ligated mares, the observed plateaux in tone and diameter prior to Day 16 or 17 and the subsequent changes were apparently not residual effects of surgical trauma followed by a gradual recovery, as the plateaux were not apparent for the ipsilateral horn of uterine-ligated mares in Experiment 2. Previous work in mares (Griffin and Ginther, 1991a) has demonstrated that changes in uterine diameter are reflective of changes in uterine tone and, as a result, monitoring diameters may be an indirect and objective means for monitoring uterine tone. The strong negative correlations between tone scores and cornual diameters obtained in the present studies support this observation. Endometrial echotexture Local effects of the conceptus on endometrial echotexture were also detected. In both experiments, the uterine segments exposed to the conceptus or

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its products had higher echotexture scores (i.e. enhanced endometrial edema and folding) than those segments without exposure (Figs. 2 and 4). The ultrasonic characteristics of the uterus are indicative of the degree of estrogen exposure (Hayes et al., 1985 ). It is possible that the observed changes in endometrial echotexture were also the result of local exposure to estrogens o f conceptus origin, as suggested for the development of uterine tone. Interestingly, in both experiments, the differences in echotexture scores for the uterine horns between segments or among groups were most apparent after Day 16 (i.e. post-fixation), indicating that intraluminally distributed products of the conceptus, and not vesicle contact per se, were responsible for the changes after fixation.

Uterine contractility In Experiment 1, there was no detectable effect of ligation on uterine contractility. This was an expected finding, as the vesicle had unlimited access to the uterine body (site of contractility assessment) in both groups. It is possible that uterine contractility was maximally stimulated in the uterine-intact group, and restriction of the conceptus to the uterine body and caudal horns was unable to elicit a further response, despite potentially greater stimulatory exposure. In Experiment 2, however, when the vesicle was restricted to one of the uterine horns, contractility in the uterine body was decreased on Days 11-14, relative to uterine-intact mares. It seems unlikely that the traumatic effects of ligation and tissue manipulation contributed to a quieting of the uterus on Days 11-14 in uterine-ligated mares, considering the average number of days (3.7 + 0.6 ) the mares had for post-operative recovery prior to daily contractility assessment and the lack of observed effect in Experiment 1. Previous work has indicated that uterine contractility is the impetus for embryo mobility (Leith and Ginther, 1985 ). Based on the present findings, the conceptus is apparently actively responsible for stimulating its own extensive mobility by stimulating uterine contractility, as has been postulated (Ginther, 1985 ). The nature of the stimulatory agents is not known. In seasonally anovulatory mares, addition of estrogen to a daily progesterone regimen did not enhance levels of contractility above those obtained with progesterone alone (Cross and Ginther, 1987). Similarly, daily administration of estrogen to early pregnant mares did not enhance contractility levels when compared with controls (Bessent et al., 1988). These results indicate that estrogens apparently are not involved in stimulating the extensive uterine contractility associated with the embryo mobility phase. It seems that the factors responsible for enhancing contractility and, therefore, mobility are distinct from those responsible for stimulating the development of turgid uterine tone a n d ultrasonically detectable edema. In this regard, Day 14 equine em-

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bryos are capable of secreting such myoactive substances as PGF, PGE2, and PGI2, in vitro (Watson and Sertich, 1989).

Corpus luteum In Experiment 2, three of six embryo losses prior to Day 25 were apparently attributable to a failure of blockage of uterine-induced luteolysis (i.e. no ultrasonically detectable corpus luteum), presumably because of restricted embryo mobility. The remaining three losses in uterine-ligated mares and both losses in uterine-intact mares occurred in mares in which the corpus luteum was ultrasonically detectable on Day 25 and, therefore, were apparently not attributable to uterine-induced luteolysis. No embryo losses occurred prior to Day 25 in Experiment 1. Work in mares (Bergfelt et al., 1989), cows (Kastelic et al., 1990), and llamas (Adams et al., 1991 ) indicates that there is a close positive relationship between ultrasonically determined luteal size and plasma progesterone concentrations, suggesting that ultrasonic assessment of the corpus luteum may be a viable alternative to plasma progesterone assay as an index of luteal function. In Experiment 1, restriction of the conceptus to the uterine body and caudal uterine horns apparently did not affect blockage of luteolysis, as the corpus luteum was ultrasonically detectable in four of five uterine-ligated mares on Day 25. In Experiment 2, the corpus luteum was ultrasonically detectable on Day 25 in eight of 10 uterine-ligated mares in which conceptus mobility was restricted to one uterine horn; pregnancy was maintained in five of the eight mares. The failure to find a significant relationship between confinement of the embryonic vesicle to one uterine horn and the frequency of luteal maintenance at Day 25 does not support previous findings (McDowell et al., 1985 ). These workers found a high incidence of luteolysis when the vesicle was confined to one horn. However, in Experiment 2, luteal diameters decreased in the uterine-ligated mares, but not in the uterine-intact mares, as indicated by a significant group by day interaction (Fig. 6). Evaluation of the relationships between restriction of the area of embryo contact and the occurrence of luteolysis was not a primary objective of the present experiments. However, these secondary observations indicate that more study is needed in this area. ACKNOWLEDGMENTS

This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison. The authors thank L. Kulick for technical assistance.

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