equine pregnancy - producci³n animal

1. Introduction

This report considers the equine intrauterine con-ceptus from the time of entry from the oviduct as ablastocyst to expulsion as a foal. A series of eventsinvolving physical interactions between the uterusand conceptus will be highlighted, and the associat-ed morphologic and physiologic aspects will be con-sidered. Most of the events were discovered andcharacterized by transrectal ultrasonic imaging andtranscervical endoscopic viewing. Certain aspects ofthe phenomena have been reviewed for embryos,1–3

fetuses,4 and both stages.5,6

The term embryo is well engrained and will beused in reference to the entire early conceptus.5 Theterms embryonic vesicle and embryo proper willalso be used when needed to emphasize or distin-guish between the entire conceptus and the fore-runner of the fetus, respectively. The embryo termswill be used only to Day 39 (Day 0=ovulation). FromDay 40 to parturition, the terms fetus or fetal stagewill be used. The choice of Day 40 as the transitionday facilitates categorizing and discussing the phe-nomena highlighted in this paper. The beginning of

umbilical cord formation, completion of replacementof the yolk sac with the allantoic sac, and the beginning of fetal activity (head nods) are onapproximately Day 40. Equine theriogenologistsand biologists should be deliberate in using theterms embryo and fetus. For example, the termsembryo mobility versus fetal mobility and embryoreduction versus fetal reduction involve distinctlydifferent mechanisms for embryos versus fetuses.

2. The Intrauterine Embryo

A. Origin of Embryonic Placental Layers

Equine theriogenologists should not consider thethree germ layers of the embryonic placenta—ecto-derm, mesoderm, and endoderm—as esoteric termsused only by embryologists. Transrectal ultrasonog-raphy has exposed veterinarians to embryonic placental membranes that formerly received littlepractical consideration. Knowledge of the layers ofthe yolk-sac wall and the membranes associatedwith transition from yolk sac to allantoic sac isrequired for full interpretation of the ultrasonic

AAEP PROCEEDINGS–/–Vol. 44–/–1998 73

MILNE LECTURE:—EQUINE PREGNANCY

Equine Pregnancy: Physical Interactions Between theUterus and Conceptus

O. J. Ginther, VMD, PhD

The equine species have evolved a series of effective, often unique, interrelated, dynamic, physicalinteractions between the uterus and conceptus. Included are embryo mobility, fixation, and orienta-tion, embryo reduction, formation of endometrial cups, fetal mobility, fetal presentation, uterine hornclosures, encasement of the fetal hind limbs by a uterine horn, and special mechanisms of uterine-fetal rotations during parturition. The morphologic and physiologic aspects of these events aredescribed. Author’s address: University of Wisconsin, Animal Health and Biomedical Sciences, 1655Linden Drive, Madison, WI 53706. E-mail: [email protected]

NOTES

Sitio Argentino de Producción Animal

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74 1998–/–Vol. 44–/–AAEP PROCEEDINGS

Fig. 1. Diagrams, sonograms, and photographs of the embryo for Days 9–15. The arrows indicate the following: Day-10 sonogram—specular reflections; Day-14 sonogram—periphery of cross-section of the uterine horn; Day-15 specimen—edge of the invading meso-derm (two arrows) and yolk-sac wall (one arrow). The Day-12 specimen shows the capsule after contraction of the blastocyst wall.Note the circular embryonic disc at Day 11 and the elongated disc at Day 15. The head is at the lower end of the Day-15 disc. Inthese and other sonograms, the graduated scale to the left is in centimeters. ed, embryonic disc.

images of singletons, differentiating singletons fromtwins, distinguishing between the membranes ofimpinging uterine cysts and embryonic vesicles, andcomprehending today’s postulates on the mecha-nisms of embryo mobility, fixation, orientation, andtwin reduction. An embryonic layer that generally isnot taught in embryology courses is the capsule,which among farm animals develops only in theequine species. Research on the anatomy of theembryonic placenta is needed because of importanceto the equine clinician and scientist. The followingdiscussion of the temporal and spatial interrelation-ships of the three embryonic layers is based on asmall number of embryonic vesicles5 and should be

considered provisional.The origin of the three germ layers and their incor-

poration into the yolk-sac wall is diagrammed forDays 9–15 (Fig. 1). Diagrams for subsequent embry-onic placental development are incorporated into thefigures for the various physiologic phenomena.

The embryo becomes a blastocyst by the time orsoon after it enters a uterine horn on Day 6.7 Theterm blastocyst is used when a central cavity forms,and the inner cell mass is established at one pole, asshown for Day 9.5 The inner cell mass will form theembryonic disc and eventually will develop into theembryo proper, fetus, and foal. The membrane sur-rounding the blastocyst cavity is a single layer of

Inner cell mass

Ectoderm (trophoblast)Mesoderm, avascularEndodermCapsule

Embryonicdisc

Yolk sac

Days 9-11

Day 11

Day 12

Day 14

Day 10

Embryonicdisc

Capsule

ed

Day 15


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ectodermal cells called the trophoblast, which contin-ues as the absorptive placental contact with theendometrium throughout pregnancy.

The capsule of an equine blastocyst developsbetween the trophoblast and zona pellucida.8 Withina day or so after the blastocyst enters the uterus, thezona pellucida is shed and the capsule becomes the outermost layer. The capsule is a thin (e.g., 3 µm)but tough, mucinous (anti-adhesive9) layer of glyco-proteins. As the blastocyst expands, the capsulethickens at least until Day 11.8 The capsule assumesconsiderable elasticity and resiliency and is a sup-portive wrapping around a delicate package duringembryo mobility (Section 2B), fixation (Section 2C),and orientation (Section 2D). It seems unlikely thatthese phenomena would have evolved in the absenceof the capsule. The capsule disappears by approxi-mately Day 21,10 suggesting that its role is completesoon after orientation. The popping sensation some-times experienced by practitioners during elimina-tion of a twin embryo by digital compression11 isattributable to rupture of the capsule.

Conversion of the single-layered wall of the blas-tocyst to a two-layered structure occurs upon encir-clement of the blastocyst cavity by a single layer ofendodermal cells. Although not studied critically,encirclement is completed by approximately Day 12.5 The resulting primitive placental vesiclecan be called a yolk sac, although some authors pre-fer to continue to call the conceptus a blastocystuntil it fixes to the uterus.10 The endoderm of theyolk sac is continuous with the endoderm of theprimitive gut (forerunner of digestive system) ofthe embryo proper. Therefore, whatever the yolksac absorbs from the intrauterine environmentbecomes available to the embryo proper. Beginninggrowth of the mesoderm from the embryonic discinto the area between the trophoblast and the yolk-sac endoderm is shown in the diagram for Day 14and by a specimen for Day 15. The invasion of themesoderm results in a three-layered wall forincreasingly greater proportions of the yolk sac, asshown on the diagrams of subsequent figures. Themesoderm differentiates into supportive connectivetissue and blood vessels.

Practitioners using an intrarectal 5-MHz ultra-sound transducer can view embryos by Days 9–11when they are 3–5 mm.6 Only about 5–10% aredetectable on Day 9, and then only those that are onthe upper end of the Day-9 range; 98% are detectableby Day 11. The vesicles are spherical and produce ablack (anechoic) circumscribed image. The sphericalshape is maintained with a progressive increase indiameter over Days 9–16. The anechoic area seen byultrasonography represents the fluid of a blastocyst(Days 9–11) or a yolk sac (Days 12–16). Althoughinvasion by the mesoderm begins by Day 14, thegerm layers and capsule are too thin to image indi-vidually. Bright white (echoic) spots often are present on the images of the upper and lower sur-faces of the blastocyst or yolk-sac parallel to the

transducer. These ultrasonically generated specularreflections12 are an aid in locating the early vesicle.For example, the Day-10 blastocyst in the sonogrammay have been missed without the specular reflec-tions. A photograph of a Day-14 conceptus is shownin the uterine body after exposure by an incisionalong the dorsal uterine midline. The Day-14 vesicleis spherical even though it is freestanding (i.e., notsubmerged in fluid), indicating that it is a turgidstructure. The uterine folds are arranged longitudi-nally, which may favor embryo mobility.

B. Embryo Mobility and Luteal Maintenance

Uterine ligation studies suggest that the embryofirst reaches the uterine body on Day 813 and thenbegins an intrauterine mobility phase that contin-ues until Days 15–17.14,15 During the embryo-mobil-ity phase, regardless of the side of ovulation, theembryo can be anywhere in the uterine lumen fromthe tips of either horn to the cervix (Fig. 2). Whenfirst detected (Days 9-11) the embryo is frequently(60%) found in the uterine body.14 Thereafter, thefrequency of entries into the uterine horns increas-es as the embryo enters the phase of maximummobility. The embryo enters every part of the uter-ine lumen and moves from one horn to another 10 to20 times per day. Practitioners must be especiallyaware of the unique embryo mobility characteristic



Fig. 2. Frontal-oblique view of suspended reproductive tract afterremoval of other abdominal viscera.

bl = bladder re = rectumbr = broad ligament rgp = rectogenital pouchinf = infundibulum ruh = right uterine hornllb = lateral ligament of bladder ub = uterine bodylo = left ovary vgp = vesicogenital pouch

luh = left uterine horn

bl

vgp

rgp

re

br

ublo

luh

llb

ruh

inf


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in this species so that the entire expanse of the uterine lumen is searched during pregnancy diagnosis. Furthermore, the mobility phenomenonis an important aspect of differentiating embryosand cysts and finding and manually eliminating amember of a twin set.

The embryo is subjected to considerable pressureby uterine contractions during embryo mobility. Somuch so, that the Day 13 or 14 vesicle may undergoperiodic compressions (e.g., every 5–14 seconds).1During compression, one dimension of the previouslyspherical vesicle can become twice as great as theother (Fig. 3). The compression phenomenon can beseen ultrasonically when the vesicle is viewed in alongitudinal section of the uterus (e.g., uterine bodywith a linear-array transducer). The widest dimen-sion of the compressed vesicle is in the longitudinaldirection of the uterine lumen. Presumably, resilien-cy and elasticity of the capsule allow uterine-induced distortions of the yolk-sac wall, but provideenough resistance against uterine contractions sothat the embryo is moved along the uterine lumen.

Data illustrating embryo mobility in a mare atDay 1316 and the extent of embryo mobility14 anduterine contractility17 on various days are shown(Fig. 4A,B). The propulsive force for embryo mobili-ty is uterine contractions as indicated by the following: (1) changes in the extent of uterine con-tractions, as assessed ultrasonically,17–20 parallelchanges in the extent of mobility (Fig. 4B);(2) mobility decreases when uterine contractionsare inhibited experimentally21; (3) contractions aregreater in parts of the uterus exposed to theembryo, based on uterine ligation studies (Fig. 4C),13 indicating that the embryo produces amyometrial stimulant that results in embryo mobil-ity1; and (4) the extent of uterine contractilitydiminishes after the embryo leaves an area.13 Inconclusion, the extensive embryo mobility isattributable to uterine contractions and is favoredby the spherical form of the vesicle, the turgidityand anti-adhesive quality of the vesicle resultingfrom the capsule (Section 2A), and the longitudinalarrangement of the uterine folds (Fig. 1).



5' 5'10' 20'

20'

25'

10'

5'

5'

5'

A

B

1614121086420

0

1

2

3

4

5

6

Number of days after ovulation

Num

ber

of lo

catio

n ch

ange

san

d sc

ore

for

cont

ract

ility

Embryo location changes

(n=7 mares)

Uterine contractility(n=13 mares)

Body Horn

Increased tone

Decreased diameter

Increased contractility

Increased edematous echotexture

Ligatures

C

Fig.4. (A) Results of a two-hour mobility trial in a single mare onDay 13. Embryo location was assigned to one of nine arbitraryuterine segments every 5 minutes. The number of minutesrequired for the embryo to move from one uterine segment toanother is shown. (B) Summary of reported data on scores for theextent of uterine contractility and embryo mobility as indicatedby the number of location changes between the uterine body anda horn during a two-hour mobility trial. (C) Local effects of theembryo on the uterus as shown by restricting the embryo to oneuterine horn.

Figure 3. Expansion and contraction of the embryonic vesicleduring embryo mobility, as viewed in a longitudinal section of theuterus.

Day 14


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In the absence of an embryo, the equine uterus pro-duces a potent luteolysin (prostaglandin F2α)22,23 thattravels to the ovary through the systemic circulation,in contrast with the more well-known local or unilat-eral route in farm ruminants.24 The indications(reviewed5,24,25) of a systemic route for uterine-induced luteolysis in mares versus a local route in cattle are listed (Table 1). In farm species, each ovaryand the major portion of the uterine tissue on that

side is drained by a common vein (uteroovarian vein;Fig. 5). In species with a local pathway (e.g., cattle),the ovarian artery is tortuous and is in close apposition to the wall of the uteroovarian vein. In a species with a systemic pathway (horses) the ovarian artery does not contact the uteroovarianvein. The presence or absence of a local utero-ovarian pathway in a given species is attributable tothese differences in vascular anatomy.



Table 1. Indicators that the Pathway from Uterus to Ovaries for Uterine-induced Luteolysisis Systemic in Mares and Local in Cows

Item Mares Cows

1. Partial hysterectomy No relationship between side of Luteal maintenance only when ipsi-removal and luteal maintenance 26 lateral uterine horn is removed 27

2. Intrauterine device 24 Stimulates luteal regression regardless Stimulates luteal regression only ifof side relationships ipsilateral to corpus luteum

3. Route of PGF2α administration No differential effect between the IU route much more effective than for inducing luteolysis 24,28,29 IU and IM routes IM route

4. Minimal luteolytic dose of PGF2α 24 Low (e.g., 5 mg) High (e.g., 25 mg)

5. Relationship of ovarian artery Artery and vein not in apposition Artery tortuous and closely appliedto uteroovarian vein 25 to vein

Fig.5. A drawing (upper-right) and photographs of cleared specimens after injection of latex into arteries (red) and veins (blue).

Mare

Cow

ua

uv

oaua

uboa oboa

oa

ov(uov)

oa

oa

ua uboaubov

Ovary

Ovary Oviduct

Uterine horn

Uterine horn

uaov

(uov)

oa = ovarian arteryoboa = ovarian branch of ovarian arteryobov = ovarian branch of ovarian vein

ov = ovarian veinua = uterine artery

uboa = uterine branch of ovarian arteryubov = uterine branch of ovarian veinuov = uteroovarian veinuv = uterine vein

oa ov (uov)ubovobov

Ovary


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Before the identity of the uterine luteolysin, com-parisons of the uteroovarian vascular anatomy ofmares (no local pathway) with that of the other farmspecies provided the earliest suggestion that thepathway between uterus and ovaries was venoarteri-al (reviewed24,25). Experimental vascular anasto-moses were used to test the hypothesis of a unilater-al venoarterial pathway without assumptions on theidentity of the luteolysin. For example, in unilateral-ly hysterectomized ewes, the uterine vein or ovarianartery from the intact side was anastomosed to thecorresponding vessel on the hysterectomized side.The anastomoses allowed luteal regression on thehysterectomized side, whereas luteal maintenanceoccurred if blood was not shunted from the intactside. Surgical anastomoses between ovarian arteriesin ewes with the donor artery originating from vari-ous levels of the vascular pedicle delineated the func-tional area of venoarterial transfer as shown (Fig. 6).

In the area of apposition between the two vessels, thevessel walls are thin and the connective tissue of theexternal layers forms a single stratum. Most likelypassage of the luteolytic substance through the wallsof the vein and artery occurs passively.

When an embryo is present, luteolysis must beblocked (first luteal response to pregnancy5)because the corpus luteum through its hormone,progesterone, is vital to embryo development.30

Evolutionary pressures apparently directedstrategies for embryo survival that were compati-ble with the systemic uteroovarian pathway foruterine-induced luteolysis in mares versus theunilateral pathway in other farm species. Themobility phenomenon in mares allows the embryoto contact all parts of the uterine lining. In thismanner, the relatively small spherical embryo is able to block luteolysis despite the relativelylarge uterus. Restricting the conceptus to a smallportion of the uterus by uterine ligation preventsdirect contact between the embryo and the remain-ing portion of the uterus and apparently results incomplete or partial luteolysis,13,31 although morestudy is needed.13 It appears that embryo mobilityevolved for blocking all of the uterus in mares,whereas expansion of the trophoblast in the uter-ine horn on the side of the corpus luteum evolvedin cattle (Fig. 7).

In addition to the reduction in exposure of the corpus luteum to PGF2α, illustrated in the diagrams,there are indications that the conceptus produces asubstance that acts as an antiluteolysin at the ovarian level. Experimental anastomoses of uterineveins or ovarian arteries in pregnant sheep and cattlehave demonstrated that the venous drainage of thegravid horn contains such a substance. (reviewed24)Studies in ewes indicate that prostaglandinE2 (PGE2) is a candidate for the antiluteolysin. Inmares, exogenous PGE2 affects uterine contractilityand tone during the time of embryo blockage ofuterine-induced luteolysis (Section 2C); a role as anantiluteolysin needs to be studied.

C. Fixation

Fixation is defined as the cessation of embryomobility.32 Discovery of the phenomena of embryomobility and fixation is a research milestonebecause it provided rationale for hypotheses on thefollowing perplexing phenomena: (1) occurrence offixation almost always in the caudal portion of oneof the uterine horns32; (2) lack of agreementbetween side of ovulation and side of fixation32;(3) more frequent embryo fixation in postpartummares in the most involuted horn33,34; (4) greaterincidence of unilateral than bilateral fixation inmares with twins, especially when the vesicles areof unequal size (Section 6A); and (5) ability of asmall conceptus to block the uterine luteolyticmechanism throughout a relatively large uterus(Section 2B).



Fig. 6. Specimen of the uteroovarian vascular stem.The two arrowsdelineate the area of venoarterial transfer of substances betweenuterine venous blood and ovarian arterial blood. Note the anasto-mosis (above upper arrow) carrying uterine venous blood into theobov in the convoluted area of the oboa.

UterusOvary

ov(uov)

ubovobov

tboa

oboaua

oa

Ewe

oa = ovarian artery tboa = tubal branch oboa = ovarian branch of ovarian artery

of ovarian artery ua = uterine arteryobov = ovarian branch ubov = uterine branch

of ovarian vein of ovarian veinov = ovarian vein uov = uteroovarian vein


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Fig. 7. Embryo-endometrial contact associated with the blockage of uterine-induced luteolysis by the embryo (luteal response to pregnancy). For illustration, the diagrams depict a single embryo and a corpus luteum (regressing=yellow; maintained=orange) ineach ovary. The mare has a systemic uteroovarian pathway; the spherical and mobile embryo covers all parts of the uterus and there-by prevents luteal exposure to PGF2α during the critical time (Days 11–15), regardless of luteal location. The cow has a unilateral pathway, and the expanding conceptus during the luteal response to pregnancy covers only the ipsilateral horn. The expan-sion of the bovine conceptus between days is shown by the sonograms (arrows) and specimens.

PGFPGF ReducedPGF

PGF

Nonpregnant Pregnant

Reduced PGFPGF

Nonpregnant Pregnant

Day 11 Day 15

Day 14

Day 13

Day 14

Day 12

Species differences inconceptus-endometrialcontact for blockage of

uterine-induced luteolysis

Cow

Mare


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Fig. 8. (A) Diagram of embryo on the day of fixation. (B) Summary of data on frequency of fixation on various days in ponies and horses.(C) Summary of data on temporal association between changing uterine tone and embryo diameter. The photographs depict changesin uterine tone. The increasing uterine tone and embryo diameter are believed to result in the cessation of mobility (fixation) at aflexure (arrows, Day 16) in a caudal uterine horn. Tone increases and confines the embryonal bulge as shown for Day 30.

On the day of fixation, the vesicle is still sphericalas shown by ultrasound studies.6 At this time, lessthan half of the yolk-sac wall has developed a thirdlayer (mesoderm), and blood vessels have begun todevelop in the mesoderm near the embryo proper(Fig. 8A).5 A cavity, the exocoelom, forms within themesoderm near the embryo proper, dividing themesoderm into two layers. Folds of ectoderm andmesoderm begin to pass over the embryo proper and

will give rise to the amnion. The membrane consist-ing of ectoderm and mesoderm is called the chorionand its future is discussed in Section 2E.

Fixation occurs on mean Days 15 in ponies and16 in horses (Fig. 8B).6 Fixation usually occurs neara flexure in the caudal portion of one of the uterinehorns. It has been postulated32 that fixation occursat this site, despite continuing uterine contrac-tions,17–19 because the flexure is the greatest

Ponies(n=47)

Horses(n=14)

181716151413

0

10

20

30

40

50

19Number of days after ovulation

Per

cent

age

of fi

xed

embr

yos

Embryodiameter

Uterine tone(score)

30

35

25

20

15

10

11 12 13 14 15 16 17 18 19

Mean day offixation

Mean day offixation

Horses(n=10)

Dia

met

er (

mm

) of

em

bryo

and

scor

e fo

r to

ne

Number of days after ovulation

Ectoderm (trophoblast)Mesoderm, avascularMesoderm, vascularEndodermCapsule

Yolk sac

Exocoelom

Chorion

A

B

C

Amniotic foldDay 3

Day 16

Day 30

Day 16


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intraluminal impediment to continued embryomobility. The photographs show the flexures in thecaudal portion of the horns for Day 16 and the corresponding location of the embryonal bulge for Day 30. The uterus rides upon and intermingleswith other viscera to a variable extent,5 and has a T-shape when lying on other viscera.

The equine uterus is flaccid during and immedi-ately after estrus as shown for Day 3, increases intone until mid-diestrus, decreases in tone until Day 10 or 11, and then, if the mare is pregnant,gradually increases in tone and becomes turgid20 asshown for Day 16. The increase in uterine tone isassociated with a decrease in uterine diameter34–36

and continues until Days 25–30 as shown for Day 30.20,37,38 The gradually increasing uterine toneand decreasing uterine diameter together withincreasing conceptus diameter6 apparently combineto result in fixation of the conceptus (Fig. 8C).32 Thishypothesis is compatible with the following: (1) ear-lier fixation and greater uterine tone in young maresthan in old mares39; (2) higher frequency of fixationin the most involuted horn postpartum33,34; (3) thelarger the embryo on Day 14, the sooner fixationoccurs18; (4) fixation when the diameter of the con-ceptus is similar to the distance between the inneropposite walls of the myometrium of the turgidhorns18; and (5) fixation a day later in horses than inponies (Fig. 8B).6 In regard to point 5, the embryo issimilar in diameter between the two mare types,6but the uterine horns are larger in diameter in hors-es.35 Fixation may be aided by a reduction in the slip-periness of the capsule9 (Section 2B).

The conceptus distributes a tone-stimulating substance during embryo mobility; uterine ligationstudies demonstrated that tone was greater in uter-ine horns exposed to the embryo (Fig. 4C).13

Estradiol may contribute to tone stimulation asindicated by the following: (1) beginning on Day 12,the conceptus produces estrogens in increasingamounts in proportion to its increasing diameter40;(2) small doses of exogenous estradiol increaseduterine tone in progesterone-primed anestrousmares37; (3) exogenous estradiol caused earlier fixa-tion and tended to increase uterine tone41; and (4) the confined conceptus locally stimulates estrus-like edematous echotexture of the endometrium.13

In regard to point 4, moderate edematous changesin the endometrium in early pregnancy do not nec-essarily indicate impending abortion. Results of arecent study42 suggest that PGE2, produced by theembryo,43 plays a role in both stimulating uterinecontractions and increasing uterine tone during theembryo-mobility phase. A continuing progesteronesource is necessary for embryo mobility and fixa-tion.30 When luteolysis occurs after fixation, theuterus loses its turgidity, and the embryo leaves thesite of fixation. Sometimes the embryonic heart isstill beating after the embryo leaves the fixation site;embryonic death, however, is imminent.44

To summarize Sections 2A–D (Fig. 9), the encap-sulated embryo stimulates myometrial contractionsand thereby travels throughout the uterus, especial-ly on Days 11–15. The mobile embryo systemicallyblocks uterine-induced luteolysis and at the sametime distributes a substance that gradually stimu-lates increasing uterine tone. By the time the block-age of luteolysis is complete (mean Day 16), thevesicle has grown and uterine tone has increased(decreased uterine diameter) to the point that fixa-tion occurs in the area of the greatest impediment tomobility (flexure in a caudal horn). This is anexquisite series of events that should pique thecuriosity of evolutionists, as well as veterinarians.

D. Orientation

Orientation is defined as rotation of the embryonicvesicle so that the embryo proper is on the ventralaspect of the yolk sac.32 The importance of orienta-tion at this time will become clear in the discussionson the transition between yolk sac and allantoic sac(Section 2E) and formation of the umbilical cord(Section 3A). The preceding diagrams (Figs. 1,8)depicted the embryonic disc on the ventral surfaceof the yolk sac. However, the mobile prefixationvesicle probably rotates, and the embryonic disc canbe anywhere on the circumference at this time. Inthis regard, simulated embryonic vesicles tend toroll when responding to uterine contractions.1

The yolk sac is three-layered and vascularized atthe embryonic pole and two-layered at the opposite

Fig. 9. Summary of events associated with embryo mobility, firstluteal response to pregnancy, and embryo fixation.

Mobility (Days 11-15)

Flexure

Fixation (Day 16)

Contraction

Embryo stimulatesuterine contractionscausing embryomobility.

1 Mobile embryodistributes a substanceto increase uterine toneand decrease uterinediameter.

3

Mobile embryo blocksuterine-induced luteolysis.

2

By the time blockage of luteolysisis completed, uterine diameterhas decreased and embryodiameter has increased sothat fixation occurs at theflexure in a caudal horn.

4


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pole (Fig. 10).5 The islands of blood cells that devel-oped in the mesoderm of the three-layered portionhave coalesced to form a continuous vascular net-work. The yolk sac does not contain stored food as ina bird egg, but with vascularization it becomes anefficient purveyor of nutritive material from theuterus to the rapidly developing embryo proper.The amniotic cavity results from a union of the twofolds of chorion that pass over the embryo as shown.Closure of the amniotic folds is completed by Day 20,10 just before loss of the capsule.

Between Days 16 (Fig. 8) and 18 (Fig. 10), much(>50%) of the yolk-sac wall is still two-layered (no mesoderm)5 as shown for Day 17 (Fig. 11). Themesoderm of the remaining three-layered wall differentiates into connective tissue and a vascularnetwork and is much stronger than the two single-cell layers of the two-layered portion. The differencein strength between the two portions of the yolk-sacwall and the relative expanse of the two portionshave been utilized to develop postulates on embryoorientation and embryo reduction in mares withtwins (Section 6A). The exposed Day-18 conceptus(Fig. 10) does not maintain a spherical shape as itdid at Day 14. The sonogram shows that in crosssections of the uterine horn the vesicles tend towarda guitar-pick or irregular shape. The apex of thevesicle is orientated dorsally, and the smooth,rounded base is orientated ventrally. The irregularshapes after fixation are normal and should not betaken as a sign of abnormal development and aharbinger of embryo loss. The shape change isattributable to uterine turgidity and thickening of the dorsal uterine wall, especially on each side of

the mesometrial attachment. The vesicle becomesmore spherical when an inhibitor of uterine tone isgiven on Day 19.45 Because of the uterine turgidity,the vesicle does not enlarge on Days 18–26 whenviewed in a cross section of the horn (Fig. 12).During this time, a compensatory increase in con-ceptus length occurs along the longitudinal uterinelumen and accommodates the increasing growth ofthe conceptus (Fig. 13).6

Uterine contractions continue after fixation,17–20

and may play a role in orientating the embryonic

Fig. 10. Diagram, sonogram, and photographs of the embryo on Day 18. The embryo proper is not yet ultrasonically detectable. Thinversus thick portions of the vesicle wall, the continuation of uterine contractions, and disproportional hypertrophy of endometrialfolds are believed to result in rotation of the vesicle after fixation. The amniotic folds have not yet closed. For color key, see Fig. 8.

Mesometrialattachment

Myometrialcontraction Endometrium

Twolayers

Threelayers

Day 18

Amniotic fold

hd

ngcp

Day 17

Fig. 11. The peripheries of mesoderm (two arrows) and yolk-sac wall(one arrow) are indicated. The embryo proper (inset) has 11 pairs ofsomites. cp, cardiac prominence; hd, head; ng, neural groove.


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vesicle. Continuous ultrasonic viewing indicates thatshape of the fixed embryo is continually altered byuterine contractions (Fig. 14).6 It has been postulat-ed32 that orientation occurs between the time of fixa-tion and the appearance of the irregular shapes.Three factors are believed1,32 to interact during orien-tation: (1) thick (three-layered) and thin (two-layered)portions of the yolk-sac wall; (2) asymmetricalencroachment from thickening of the upper turgid

uterine wall on each side of the mesometrial attach-ment (Fig. 15); and (3) the massaging action of uterinecontractions. The interaction of these factors results in the thickest portion of the yolk-sac wall(embryonic pole) rotating to a ventral position(antimesometrial). It is not clear, however, whetherthe localized thickening of the endometrium adjacentto the thin-walled portion of the yolk sac (Fig. 10)occurs before, during, or after orientation; the cause ofthe thickening has not been determined. The orienta-tion hypothesis is compatible with six reported casesof disorientation associated with a flaccid uterus orthe presence of twin vesicles at the postulated time oforientation.1,6 Once fixation and orientation areestablished, increasing horn turgidity, cranial andcaudal to the vesicle (Fig. 8), prevents the vesicle fromdislodging longitudinally in the uterine lumen. Theorientated position, as viewed in cross-section, appar-ently is aided by adhesiveness and cross-ridging ofthe endometrial folds.10 Later, after loss of the capsuleon Day 21 (Section 2A), a ridge of trophoblast indentsthe endometrium and may further anchor the vesicleand help prevent rotation.10

Fig. 13. Cross and longitudinal sections of an embryonic vesicle.The longitudinal section shows that expansion occurs along theuterine lumen and accounts for the plateau in cross-sectionalexpansion on Days 18–26 that is shown in Fig. 12.

Fig. 14. Example of uterine massage of the vesicle on Day 18.The vesicle changed shape when viewed continuously. The twosonograms were taken at an interval of 3 seconds.

Fig. 15. Cross-sectional views showing the disproportional hyper-trophy of the dorsal endometrial folds after Day 17. The outerlimits of the uterine wall are delineated by arrows.

Fig. 12. Cross-sectional growth profile of the embryonic vesicle.

9 13 17 21 25 29 33 37

0

8

16

24

32

40

48

56

Mea

n of

hei

ght a

nd w

idth

(m

m)

Days11-16

Days 16-28 Days 28-39

11 15 19 23 27 31 35 39

4

12

20

28

36

44

52

Number of days from ovulation

Horse embryos(n=9 to 20/day)

Day 18Cross section

Longitudinalsection

Day 18 Sec 0 Sec 3

Day 17 Day 22


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Fig. 16. In the photographs of the embryonic vesicles at Days 21 and 24, the vesicles were submerged and tilted slightly to exposethe embryonic pole. The broken line for Day 21 connects the embryo propers among illustrations, and the line for Day 24 connectsthe allantoic sacs. Note the emergence and position assumed by the allantois. Lengthening of the limb buds between Days 25 and 26is apparent. The genital tubercle (forerunner of clitoris and penis) is already visible at Day 26 and by Day 60 will have migrated toa position that will allow ultrasonic diagnosis of fetal gender.as = allantoic sac fl = forelimb hl = hind limb mb = mid brain tl = tailfc = future cerebrum fmo = future medulla oblongata ht = heart pa = pharyngeal arches

fcl = future cerebellum gt = genital tubercle ma = mandibular arch pf = pontine flexure

E. Transition from Yolk Sac to Allantoic Sac

By Day 21, the amniotic cavity is completelyformed (Fig 16). The allantois has begun to emergefrom the hindgut as shown in the diagram and pho-tograph of a Day-21 embryo proper. The allantois

grows into the exocoelom.5 The allantois becomesprominent by Day 24, as shown in the sonogram andphotograph. In the sonogram, the allantoic membraneand the closely apposed yolk-sac membrane appear asa thin echoic line suspending the embryo proper.

Ectoderm (trophoblast)Mesoderm, avascularMesoderm, vascularEndoderm

Yolk sac

Amniotic sac

Allantoic sac

Chorion

Allanto-chorion

Day 21

Day 24

Day 20 Day 21 Day 25 Day 26

ht

as fl hl

pa

pf

fl

ht

mbfc fcl

fmo

tl

ma

hl gt

fl


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equine pregnancy - producci³n animal

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