Hopefully if the exact answer is not clear at the end of this lecture, there will at least have been a useful discussion of all these queries.

Pregnancy in the Mare: General Physiology

Fertilisation occurs in the ampulla of the oviduct; enough sperm are present within 4 hours

Tubal transport lasts 5 to 6 days; the oviduct retains unfertilised ova; mechanism?

MOBILITY phase in uterine horns and body until Day 16: FIXATION (termination of mobility) then occurs. Fixation should be at the base of one of the uterine horns. This early mobility of the equine conceptus is important to block endometrial prostaglandin release. The prostaglandin arrives via a systemic pathway as the mare does not have a close connection of the uterine vein and ovarian artery

IMPLANTATION begins around Day 40 and is not complete until around Day 140 when formation of the micro-cotyledons is complete. Attachment of the placenta is gradual in the mare and is unable to be precisely temporally defined

ENDOMETRIAL CUPS: Distinctive, irregular-shaped areas which develop to become grossly visible arranged in a ring at the base of the pregnant horn. Begin to produce equine chorionic gonadotrophin (eCG; old term PMSG) and appears in the circulation around Day 35. Values rise rapidly to peak around 65 Days. There is then a variable rate of decline, but may persist as long as 150 days. This effectively means the mare cannot be bred again that year once she is positive for eCG. eCG is responsible for ovulation and/or luteinisation of secondary ovulations.

Progesterone Sources: Until Day 200: primary and secondary corpora lutea

From Day 60 : Feto-Placental Unit produces progestins and gradually assumes the main role

Uterine changes: becomes tonic (17 -21 Days) and swelling develops about 21 - 24 Days; by 30 Days the embryonic vesicle is 3 to 4 cm in diameter and by 40 Days is the size of a tennis ball; between Days 60 and 100, the uterus is low within the abdomen and the fetus can not usually be palpated; from 4 to 5 months onwards the fetus can usually be palpated.

Cervix changes: tight, firm and tonic (as during dioestrus)

Ovarian changes: 18 to 40 Days - many follicles up to 3 Cm, occasional ovulations; 40 to 120 Days - extensive ovarian activity with ovulations; luteinisation and development of secondary corpora lutea; 120 days to term: small and inactive, difficult to palpate after 5 months duration of pregnancy is 330 to 345 Days, but enormous variation is possible and anywhere from 315 to 360 Days is frequently reported .

Diagnosis of early pregnancy using ultrasound: Day 11

The equine embryonic vesicle can be reliably detected at day 11 when sufficient anechoic yolk sac fluid has developed.

The black yolk sac fluid is enclosed within the trophoblast resulting in a spherical outline with a diameter of 5 to 8 mm.

Note the two hyperechoic short lines on the dorsal and ventral borders of the conceptus known as specular reflections. Whilst they may aid location of the early embryonic vesicle, they are not indicative of pregnancy but are a physical phenomenon arising from the reflection of the ultrasound beam; cysts tend to cause non-specular reflections.

The anechoic vesicle is highly mobile at this stage and found in either horn or in the body. It is, therefore, important to examine both uterine horns and the body thoroughly.

Mares are not usually scanned as early as Day 11 because it is possible to miss the conceptus if scanning conditions are not ideal and the ovulation date is not accurately known. If there was an ovulation one or two days after the first ovulation, any pregnancy arising from this later ovulation would be too small to be detected.

Diagnosis of early pregnancy using ultrasound: Day 14

The 14 day conceptus is 13 to 18 mm in size and lies centrally in the uterine body.

Note the spherical shape and increase in size over the Day 11 pregnancy. The embryonic vesicle grows at a rate of approximately 3.5 mm/day at this early stage of pregnancy and remains highly mobile, making thorough examination of all parts of the uterus important.

In the event of twin pregnancies, both vesicles can usually be seen at 14 days, even if the second co-twin arose from a later ovulation.

This fact, together with the mobility and relatively small size of the concepti make 14 to 15 days the optimal stage of pregnancy to diagnose twins and crush one co-twin.

Although pregnancy diagnosis is highly accurate even at this early stage, it is important to be aware of the possible confusion caused by uterine cysts and the presence of twin conceptuses. Ideally one would have performed an ultrasound examination before breeding the mare, but this is not always possible. If the first scan is performed at Day 14 or 15, then it is possible to return the next day in cases of confusion and see if the pregnancy has changed position or grown in size. This should allow differentiation from a cyst before the pregnancies have a chance to become unilaterally fixed.

Another advantage of performing the first examination for pregnancy at Day 15 and not Day 18 is that a mare with a shortened luteal phase due to endometritis can be detected. If examination is delayed until Day 18 it is possible that the mare has ovulated already and the fact that she has had a shortened luteal phase will not be detected.

Many veterinarians point out to me that it may be better to delay the first examination until Day 18 when non-pregnant mares have had the opportunity to return to oestrus and it may be possible to predict the next breeding time. However it will be very difficult to correct unilateral twins. Whilst there is undoubtedly a natural reduction mechanism of twin pregnancies to singletons in mares, I believe that the effectiveness of this has been exaggerated and is not as successful as manual crushing of one twin at Day 15 which has a 95% success rate in my experience.

Double ovulations occur during 8 to 30% of oestrous cycles, the frequency depending upon the breed and type of the mare (Thoroughbreds, highest rate; ponies, lowest rate). Accurate detection of such ovulations is important as twinning is undesirable, firstly because it accounts for 10--30% of abortions and, secondly, even if both fetuses survive and are carried to term, many are dysmature, resulting in a high neonatal mortality rate. A further complication is that if embryonic/fetal death occurs after the formation of the endometrial cups these latter structures persist until they spontaneously regress as if pregnancy had been maintained, resulting in pseudopregnancy.

Effective twin management is a vital part of maximising fertility on the stud farm. In my experience owners have accepted the early pregnancy check when the reasoning has been explained to them. Of course, if the mare must be transported back to the stud farm or to the veterinarian for scanning this may present more of a difficulty.

It is important to realise that if twin pregnancies arise from asynchronous ovulations, once conceptus is likely to be larger than the other. This is not an indication to leave the smaller pregnancy, manual crushing should still be performed

By day 16 of pregnancy the vesicle is normally fixed at the base of either the left or the right horn. The shape is still regular, but more ovoid than strictly spherical.

At day 20 of pregnancy the conceptus is irregular in outline. This irregular shape is normal and is not an indication of imminent pregnancy loss. The appearance can be so irregular that it could be confused with a collection of intraluminal uterine fluid, particularly as no embryo is likely to be visible at this stage. Therefore, a single pregnancy scan at Day 20 is a bad idea. Note the thickened dorsal wall of the uterus and the presence of a slight oedema pattern above the conceptus. This may be due to oestrogen production by the conceptus or the follicular development typical of this stage of pregnancy. If the pregnancy is normal, the primary corpus luteum should also be visible. If the oedema pattern of the uterus becomes widespread it may be an indicator of early embryonic death (see later). It is important to re-schedule subsequent pregnancy examinations at more frequent intervals than normal and the first re-examination should be in 3 or 4 days to monitor the pregnancy.

By day 24 of pregnancy, the embryo is approximately 6 mm in length. The heartbeat can normally be detected as a flickering movement in the middle of the echoic embryo around this stage of pregnancy. Note the emergence of the allantoic sac as a small anechoic area from beneath the embryo. Over the next few days, the development of the allantois will lift the embryo dorsally and the yolk sac will gradually reduce in size. This change in ratio of the yolk sac to allantois is an important feature in ageing pregnancies. It is important to recognise the embryo and identify a heart-beat because the irregular shape of the vesicle is easily confused with an endometrial cyst.

In the day 28 pregnancy note the developing allantois, the regressing yolk sac and the associated dorsal 'ascent' of the embryo.The apposition of yolk sac and allantois results in an ultrasonically visible thin line normally orientated horizontally. The embryo is visible as an echoic mass on this line.

By day 30 of pregnancy note the enlargement of the allantoic sac such that the two sacs are approximately equal in size. The embryo is highly echogenic and is visible on the line separating the allantoic and yolk sacs and the heartbeat can be clearly seen.

By day 33 the embryo is usually in the dorsal part of the vesicle and is approximately 14 mm in length. The volume of the allantois greatly exceeds that of the yolk sac.

Pregnancy Diagnosis: Protocol (When Ovulation Time is Known) double ovulation single ovulation

• first exam Day 14 to 15 Day 17 to 18
• second exam Day 24 to 27
• third exam Day 33 to 35
• autumn exam October
• (day of ovulation is Day 0)

Following an initial examination at Day 15, the aim of the examination at Day 24 to 27 should be to assess the embryo is developing normally (increase in size, normal echogenicity of the yolk sac and so on) and identify the heartbeat. In addition it can be conformed that there is only a single conceptus. If twins were inadvertently missed at the earlier examination, it may still be possible to correctly manage them.

Ideally a third examination should be performed around Day 33 to 35. The aim of this examination is to confirm that a single conceptus is developing normally. If there is failure of normal development or if twins are detected, it is usually possible to terminate the pregnancy and re- breed the mare.

If examination is delayed until after day 33, the endometrial cups may have developed and even if pregnancy is terminated, eCG production may continue for a variable time, sometimes preventing normal oestrous cycles for the rest of the breeding season. In addition much time may be lost if early embryonic death has occurred and earlier scanning will allow earlier detection of this loss.

Twin Pregnancy
Undesirable: abortion often at 7 to 8 months or undersized or dead foals at term. The reason for the low survival rate of twins is due to competition for placental space. Recent studies using transrectal ultrasound imaging has shown that the mare has an embryo reduction mechanism so that there is a wide disparity between the number of double ovulations and twin births. Most embryo reduction occurs after fixation at day 17, and it is greatest when it occurs in the same horn and when the conceptuses are of unequal size. A deprivation hypothesis has been suggested.

Almost always dizygotic (but see Meadows et al (1995); Equine Vet. J. , 27, 394-397)

Important to recognise double ovulation and so examination of the ovaries is important at every examination for pregnancy to provide information on the number and appearance of the corpora lutea. Mares with twins almost always have two corpora lutea.

Double ovulations can be synchronous or asynchronous; primary or secondary. Where the second ovulations is more than 72 hours after the first, a pregnancy is unlikely to result. Mares should not normally be re-bred after an ovulation has happened twins can undergo unilateral or bilateral fixation spontaneous embryo reduction: the natural reduction of one member of a set of twins active embryo reduction: crushing

Management of Twin Pregnancy in the Mare
The management of multiple conceptuses depends on the time of detection. An important consideration of the clinician is the reduction of conceptus number before the production of eCG (or, less satisfactorily, the termination of the pregnancy)

Imaging bilateral multiple conceptuses + unilateral separate conceptuses is uncomplicated

Bilateral twins 14/15 Days after ovulation: manage by manipulating the smaller conceptus using the transducer (or fingers) towards the tip of the uterine horn or the cervix and then crush the conceptus against these structures. Where the conceptual vesicles are of dissimilar size, usually the smaller conceptus should be ruptured. This is easier at Days 14 to 15 when they are 14 to 20 mm in diameter than Days 11 to 13 when they are 6 to 11 mm in diameter. No flunixin (Banamine) or progesterone administered. The author prefers to gently separate the conceptual vesicles using the transducer to enable the procedure to be imaged. Pressure is put on the selected conceptus using the transducer once they have been separated. The spherical shape is lost and then a distinct popping sensation is felt. The small amount of fluid is rapidly resorbed and the remaining conceptus should look normal, although there may initially be a small amount of fluid form the crushed conceptus around it. Occasionally uterine relaxant may facilitate the procedure.

When unilateral multiple conceptuses are adjacently positioned the diagnosis may be less straightforward

Multiple conceptuses 17 Days after ovulation

Bilateral: crush up until Day 32 or even later

Unilateral: attempt to crush one or the pregnancy can be terminated by use of PGF2alpha or leave both until 25/30 days which allows time for any natural embryo reduction to take place, but is before production of eCG

If no reduction consider twin puncture (see article in The Horse). Position one conceptus under the puncture line of the scanner by rectal palpation, then puncture the conceptus and remove the fluid

management of twin pregnancies after this period is complicated by the formation of endometrial cups and if twin pregnancies are not successfully managed before the cups are formed or both embryos die after day 37, the mare usually will not return to a fertile oestrus for a prolonged period of time.

Methods of managing twins after day 37 of gestation are variable and unreliable. Dietary energy restriction, surgical removal of one vesicle, intracardiac injection of potassium chloride using transabdominal ultrasound and, most recently, transvaginal ultrasound-guided needle-puncture have all been attempted. The latter would appear to offer the best approach and has been successfully used by the author on several occasions.

Although accurate interpretation of the ultrasound image of early pregnancies in the mare and the technique of crushing a conceptus are skills which need experience, the advent of B-mode ultrasound imaging has provided a method of more readily managing twin pregnancy in the mare. Consequently, multiple ovulation in the mare should not be regarded as a reason for withholding mating. In fact, pregnancy rates are improved after twin ovulation.

Early Embryonic Death in the Mare

Pregnancy failure
Pregnancy failure is a source of major economic loss to the horse industry. Embryonic death occurs before 40 days of gestation when organogenesis is complete, with early embryonic death (EED) occurring before the maternal recognition of pregnancy. Early fetal death occurs before 150 days of gestation; thus, late fetal death occurs afterwards. Abortion is defined as expulsion of the fetus and its membranes before 300 days, whereas a stillbirth is expulsion of the fetus and its membranes from day 300 onwards.

Early embryonic death
In normal fertile mares the fertilization rate is over 90%, which is comparable with other domestic species, with estimates of the EED rate at between 5 and 24%. In subfertile mares, the rate is higher. The differences in the estimates are due to varying methods of pregnancy detection, and the animals studied. The period of greatest embryonic death in subfertile mares occurs in the interval before pregnancy can be detected with ultrasound (day 11), particularly at the time the embryo enters the uterus. Between days 14 and 40, the rate of embryonic death varies between 5% and 15%.

Early embryonic death: causes
EED is multifactorial, in which external factors such as the environment and management as well as pathophysiological factors are involved. However, the evidence for many of these associations are anecdotal and frequently contradictory.

Maternal Factors

A number of maternal factors including hormone deficiencies and imbalance, uterine environment, age and lactation have been implicated.

Hormonal deficiencies and imbalance

Progesterone is critical for the maintenance of pregnancy in mares. The only source of progesterone during the embryonic period is the primary corpus luteum. On the assumption that luteal insufficiency is important in EED, many mares are given exogenous progesterone or progestogens in an attempt to prevent it occurring. However, the rationale for this widespread practice is highly questionable, although primary luteal insufficiency as a cause of EED has been reported by Bergfelt et al. (1992; J. Reprod. Fert. 95, 339). Progesterone supplementation has been recently reviewed by 'Twink 'Allen (1993; Equine Vet. J. 25, 90), who is sceptical of any benefit. Many dosage regimens used do not effectively elevate or maintain plasma progesterone levels. Withdrawal of supplementary progesterone therapy during midgestation may leave the clinician open to criticism if the mare subsequently aborts. In the author's opinion, progesterone therapy is most appropriate in mares which have uterine oedema and an indistinct corpus luteum at the time of first examination for pregnancy (15 days).

Uterine environment

An abnormal uterine environment is detrimental to embryonic survival. Endometritis may result in EED by inducing premature luteolysis or because of its direct effect on the embryo. Ideally, it would be preferable to avoid mating mares with acute endometritis; however, with the development of more effective treatments for postmating endometritis, in certain cases it may be preferable to mate mares even if there is evidence of the acute disease.

Severe periglandular fibrosis of the uterine glands may reduce the chances of embryo survival. Not only is this a response to persistent endometritis, but it also increases with age. This is one of the reasons for the reduced fertility of mares over 12 years of age, for despite similar fertilization rates, detected pregnancy rates are on average 33% lower. Paradoxically, increased embryonic death is occasionally seen in mares that are bred at a young age (11--16 months); the reasons for this are not known.

The mare normally resumes cyclical ovarian activity very shortly after parturition so that they are sometimes served as early as 7--10 days postpartum (at the foal heat). There is conflicting evidence about the level of embryonic death if fertilization occurs at this time, with some studies showing a higher rate and others no effect. A clear advantage of breeding at the first oestrus postpartum is that the foaling to conception interval is significantly shorter; 25 versus 44 days has been reported.

The reasons for the apparent decreased fertility in mares mated at the foal heat is the hostile uterine environment due to delayed uterine involution or persistent endometritis. However, pregnancy rates are clearly influenced by how strict the selection criteria are for mating at the foal heat. Traditionally, such factors as a normal foaling, placental expulsion, minimal vaginal bruising and absence of infection have been used. Endometrial cytology and ultrasonic scanning of the genital tract of each mare may be more reliable methods on which to base a decision.

Lactation

More pregnancy failures are detected in lactating than non-lactating (maiden or barren) mares; this also increases with the age of the mare.

Embryonic Factors

Embryonic abnormalities are also important to consider in relation to embryonic death. Embryos recovered from subfertile mares are smaller and have more morphological defects than embryos from fertile mares; however, this may be due to an abnormal uterine environment. Ultrasonic scanning has provided a valuable tool in studying embryonic death. Because pregnancy is often diagnosed at an early stage, it is important to inform owners that not all pregnancies detected with ultrasound will survive, even in apparently normal mares. There are certain morphological features detected with ultrasound that are typical of mares in which embryonic death is occurring. Some of the consistent features include: (1) presence of fluid within the uterine lumen; (2) prominent endometrial oedema; (3) decreased or prolonged conceptus mobility; (4) undersized or irregularly shaped conceptus; (5) cessation of embryonic heart beat; (6) reduced volume of placental fluids; (7) disorganization of placental membranes; (8) hyperechogenic areas in the embryo and membranes.

Fetal Death/Abortion

An overall abortion rate after 60 days of gestation of 10% is usually cited for the horse. In practice, it is important to distinguish infectious from non-infectious causes. Vaginal discharge, premature lactation and colic in pregnant mares may indicate an impending or recent abortion. Whatever the suspected cause, when abortion occurs, the mare should be isolated, a history obtained and the fetus sent to an approved laboratory for autopsy. If a veterinary surgeon wishes to perform a post-mortem examination, small but representative samples of liver, lung, thymus, spleen and chorioallantois (two samples, one of which is from the cervical star, which is the irregular, star-shaped avillous area of the chorion that lies over the internal os of the cervix) should be sent in formal saline for histological examination. In addition, frozen samples of fresh fetal liver and lung should be stored in a deep freeze should viral isolation investigation be required at a later stage. Paired serum samples from the mare and close companions should also be taken for serological investigation. Swabs from fetal heart or liver and the cervical pole of the chorion are used to screen for bacterial infection. The fetus and fetal membranes (amnion, chorioallantois and umbilical cord) must be carefully examined for the presence of abnormalities and areas of discoloration.

The causes of equine abortion can be broadly divided into non-infectious (70%), infectious (15%) and unknown (15%).

Non-infectious Causes of Abortion and Stillbirth

Twinning

Historically, twins have been the single most important cause of abortion in Thoroughbreds. However, it is now much less common due to the widespread use of ultrasonography. The diagnosis of twin pregnancy can be made even if only one fetus is found, as examination of the placenta reveals an area devoid of villi where the two placentas were in contact. Twins should still be submitted to a diagnostic laboratory as twin pregnancies are not protected from equine herpes virus (EHV) infection.

Umbilical cord abnormalities

In mares, the umbilical cord is twisted, usually in a clockwise spiral. The normal length ranges from 36 to 83 cm (mean 55 cm). Increased cord length has been associated with excessive cord torsion, which can cause vascular obstruction of the urachus. This can result in abortion of an autolysed fetus. Decreased cord length can cause premature tearing of fetal membranes, leading to fetal asphyxia.

Premature placental separation

In mares, the interdigitating microvilli are connected by an unidentified electron-dense material. Placental separation involves dissolution of this substance. Causes of premature placental separation are largely unknown although maternal stress and endophyte-contaminated tall fescue have been implicated. When placental separation occurs shortly before parturition, the thickened placenta often does not rupture through the cervical star, and the allantochorion bulges out of the vulva (`red-bag' delivery). The foal can become hypoxic, resulting in the neonatal maladjustment syndrome.

Body pregnancy

In this condition almost the entire chorionic surface of the placenta contained within the uterine body is without villi, while that contained within the horns is covered with an excessive number of villi. The proportion of the placenta corresponding to the two uterine horns is small, and the fetus is situated entirely within the uterine body. The fetus is frequently aborted completely contained within its placenta; its growth has been retarded. The abortion occurs when the nutritional demands of the fetus exceed the ability of the placenta to meet them.

Fetal abnormalities

Severe developmental anomalies involving the central nervous system or development of body cavities have been reported in aborted fetuses.

Maternal disease

Pyrexia, and malnutrition during pregnancy, have been implicated as causes of abortion.

Sound Track for The Mare in Vision: Video on Equine Pregnancy Detection

Diagnostic ultrasound plays a pivotal role in the reproductive management of the mare. Consequently a thorough understanding of normal ultrasonographic anatomy is vital for veterinarians involved in broodmare work.

This video-tape presents a complete guide to the use of transrectal ultrasonography for early pregnancy detection in mares. Ultrasonographic images are shown both free from annotation and with explanatory symbols. A full soundtrack describes the key features of each image.

Normal equine pregnancies during the first 60 days of gestation and how these findings relate to the management of pregnancy are shown. Variations in the appearance of normal anatomy, ovarian features of early pregnancy, early embryonic death, endometrial cysts and twins are also presented.

To carry out examinations safely, mares should be suitably restrained. Ideally, one should have a set of stocks approximately 75 cm (30 inches) wide and just longer than an average mare. This is adequate for most animals and will even accommodate large draught mares.

In a few cases, a twitch may be required to provide additional restraint.

Foals should be restrained in front of, or to the side of the mare.

Tying the tail to one side keeps it out of the way, clean and prevents hairs entering the rectum.

Precautions necessary for rectal examinations also apply to ultrasound examinations and rectal examination should always precede the ultrasound examination.

An initial rectal examination ensures removal of all faecal material, facilitates rapid location of the reproductive tract during scanning and provides information on texture of structures.

The scanner should be as close to eye level as practicable and the control panel of the machine within easy reach of the operator. The scanner can be placed either side of the mare. Where the operator's left hand holds the transducer, the scanner is placed obliquely to the right side of the mare's hind-quarters allowing the right hand to make any notes or adjustments to the controls.

To facilitate correct orientation of the transducer, a groove for the finger of the operator is usually located on the transducer, on the opposite side to the working face.

The fingers should always be in front of the transducer as it is being introduced and later manipulated rather than pushing the transducer on ahead.

For reasons of hygiene, it may be desirable to have the transducer in a plastic sleeve. Coupling gel should be used to exclude air from between the transducer and its protective cover.

Using copious amounts of lubricant, which also acts as a coupling medium to ensure good contact and prevent air interference, the transducer and hand are gently inserted into the rectum. Should the mare strain, the examination should be stopped and one should wait for the rectum to relax. However, straining is usually not a significant problem.

It is best to examine the reproductive tract systematically and to scan the entire uterus and both ovaries at least twice.

In this graphic, the transducer is held within the rectum in the longitudinal plane. Since the uterus of the mare is 'T-shaped', the uterine body appears as a rectangular image in the longitudinal plane. When scanning the uterine body, it is important to move the transducer forwards and backwards and from side to side so that no feature is missed.

It is important to move the transducer slowly at all times.

To image the uterine horns and ovaries the transducer should be rotated slowly to the right and then the left side. Therefore, the uterine horns appear as circular images in cross-section.

If difficulties are encountered with finding a structure, the transducer can be withdrawn a short distance and the structure located by palpation. Ultrasound examination can then be resumed.

The subsequent images were taken with a Pie Medical Scanner 200-Vet with a 5 and 7.5 MHz dual-frequency linear-array transducer. The 5 MHz frequency was used in this series of images.

In all ultrasound images on this tape the cervix would be to the left of the image.

The day of ovulation is designated Day 0 and is the first day that a large follicle observed the previous day is no longer present.

The equine embryonic vesicle can be reliably detected at day 11 when sufficient anechoic yolk sac fluid has developed. This is the age of the vesicle in this image.

The black yolk sac fluid is enclosed within the trophoblast resulting in a spherical outline with a diameter of 6 mm.

The anechoic vesicle is highly mobile at this stage and can be found in either horn or in the body. It is, therefore, important to examine both uterine horns and the body thoroughly.

Note the two hyperechoic short lines on the dorsal and ventral borders of the conceptus. These artifacts are known as specular reflections. While they may be helpful in locating the early vesicle, neither indicates the presence of an embryonic disc as was once thought.

This 14 day conceptus is 15 mm in size and lies centrally in the uterine body. Note the spherical shape and increase in size over the day 11 conceptus. The embryonic vesicle grows at a rate of approximately 3.5 mm/day at this early stage of pregnancy and remains highly mobile, making thorough examination of all parts of the uterus important.

Specular reflections can again be seen.

The broad column of relatively brighter appearance immediately beneath the vesicle is due to an enhancement artifact.

In the event of twin pregnancies, both vesicles can usually be seen at 14 days, even if the second co-twin arose from a later ovulation. This fact, together with the mobility and relatively small size of the concepti make 14 to 15 days the optimal stage of pregnancy to diagnose twins and crush one co-twin.

This 15 day conceptus is almost 20 mm in diameter.

The vesicle remains spherical and mobile and still has a rapid growth rate, useful in differentiating it from a cyst.

By day 16 of pregnancy the vesicle is normally fixed at the base of either the left or the right horn.

The shape is still regular, but more ovoid than strictly spherical.

At day 20 of pregnancy the conceptus is irregular in outline. This irregular shape is normal and is not an indication of imminent pregnancy loss. The appearance can be so irregular that it could be confused with a collection of intraluminal uterine fluid.

Note the thickened dorsal wall of the uterus and also the presence of a slight oedema pattern above the conceptus. This may be due to oestrogen production by the conceptus or the follicular development typical of this stage of pregnancy. If the pregnancy is normal, the primary corpus luteum should also be visible.

If the oedema pattern of the uterus becomes widespread it may be an indicator of early embryonic death. It is important to re-schedule subsequent pregnancy examinations at more frequent intervals than normal and the first re-examination should be in 3 or 4 days to monitor the pregnancy.

This image is also of a day 20 pregnancy. As expected, the conceptus is irregular in outline, but here the embryo is seen as a 3 mm, echoic oval structure found in its normal site ventrally within the trophoblast vesicle. Rarely the embryo is first detected at a different site and whilst the reason for this is not known, such pregnancies appear to develop normally.

The detection of an embryo in this image but not the previous 20 day image indicates that some variation in appearance of features is normal. In addition, if the mare was examined daily during breeding, the true age of the conceptus can vary within a range of 0 to 24 hours. This alone may account for considerable variation in appearance of embryos at known gestation lengths. However, all embryos should be detectable by day 24. Failure to detect an embryo after that time is an indication that the pregnancy will ultimately fail.

By day 22 the embryonic mass is 4 to 5 mm in size although for the vesicle there is typically a plateau in growth rate between days 18 to 26.

It is important to have an accurate history of the age of the pregnancy because small for age vesicles can indicate impending early embryonic death. From this stage of pregnancy onward, morphological changes are also used to age the pregnancy. These include shape changes, the location of the embryo within the trophoblast and the relative sizes of the yolk sac and allantois.

In this image of a day 24 pregnancy, the embryo is approximately 6 mm in length. The heartbeat can normally be detected as a flickering movement in the middle of the echoic embryo around this stage of pregnancy.

Note the emergence of the allantoic sac as a small anechoic area from beneath the embryo.

Over the next few days, the development of the allantois will lift the embryo dorsally and the yolk sac will gradually reduce in size. This change in ratio of the yolk sac to allantois is an important feature in ageing pregnancies.

It is important to recognise the embryo and identify a heart-beat because the irregular shape of the vesicle is easily confused with an endometrial cyst.

In this day 28 pregnancy the developing allantois, the regressing yolk sac and the associated dorsal 'ascent' of the embryo should be noted.

The apposition of yolk sac and allantois results in an ultrasonically visible thin line normally orientated horizontally.

The embryo is visible as an echoic mass on this line.

This day 28 pregnancy illustrates that the echoic line which represents the apposed walls of the yolk sac and allantois can sometimes be aligned vertically rather than horizontally in normal pregnancies. However, caution is advised because this appearance can also be confused with twin pregnancies of earlier gestation when the breeding history is unknown.

In this image the yolk sac is to the left and the allantois to the right.

By day 30 of pregnancy note the enlargement of the allantoic sac such that the two sacs are approximately equal in size.

The embryo is highly echogenic and is visible on the line separating the allantoic and yolk sacs.

In this day 33 pregnancy the heartbeat can be clearly seen.

At day 33 of pregnancy the embryo is usually in the dorsal part of the vesicle, in this case at a "2 o'clock" position. The embryo is approximately 14 mm in length.

The volume of the allantois greatly exceeds that of the yolk sac. The aim of this examination is to confirm that a single conceptus is developing normally. If there is failure of normal development or if twins are detected, it is usually possible to terminate the pregnancy and re-breed the mare. However, if examination is delayed until after day 33, the endometrial cups may have developed and even if pregnancy is terminated, eCG production may continue for a variable time, sometimes preventing normal oestrous cycles for the rest of the breeding season.

This image, also of a 33 day pregnancy illustrates the variation that can occur in the location of the embryo. The embryo is again high in the dorsal part of the vesicle, but in a "10 O'clock" position.

Such variations in appearance of the image are to be expected and arise due to natural variation or can even be caused by the location of the transducer within the rectum.

By day 38 of pregnancy the allantois surrounds the yolk sac which has almost disappeared. The allantoic membranes are nearly opposed.

The embryo is approximately 17 mm in length and as seen here, is usually found on the mesometrial side of the trophoblastic vesicle.

By day 43 the replacement of the yolk sac by the allantochorion placenta is almost complete and the membranes of the allantois have converged on it to form the umbilical cord, clearly visible in this image.

The yolk sac is still visible as a small anechoic area within the developing umbilical cord.

As the foetus grows it descends from its position proximal to the mesometrium and the umbilicus lengthens accordingly.

By day 45, the foetus has descended approximately two-thirds of the way towards the ventral part of the allantois.

The developing head of the foetus is recognisable.

By day 60 of pregnancy the diameter of the conceptus exceeds the scanning width of the ultrasound probe.

Considerable foetal motility is obvious.

This day 31 pregnancy is developing normally. Examination of the ovaries is important at every examination for pregnancy to provide information on the number and appearance of the corpora lutea. Mares with twins almost always have two corpora lutea. The primary corpus luteum is visible as a uniformly echoic area on this image. A corpus luteum readily detectable on ultrasound is usually producing progesterone. Ultrasonographic assessment of the corpus luteum may be useful in assisting a decision to provide exogenous progesterone or progestogen supplementation.

Note the presence of several follicles which is quite normal for early pregnant mares.

During the examination of pregnant mares between 40 and approximately 150 days, large follicles and supplementary corpora lutea are frequently found in addition to the primary corpus luteum.

As in this image of a mare 48 days pregnant, the supplementary corpora lutea are often as large as 6 to 8 centimetres, with thick echoic walls and anechoic centres cris-crossed by echoic trabeculae.

Uterine cysts are fluid-filled and are immobile, anechoic structures with an obvious border.

They can be located anywhere in the uterus and, whilst their precise relationship to subfertility is not clear, they are the structures most likely to confuse the diagnosis of early pregnancy.

Differentiation is easiest based on previous mapping of cysts before the mare has been bred and is also possible based on the characteristic mobility and rapid growth rate of the early pregnancy.

Spherical-shaped cysts such as these remain difficult to distinguish and previous cyst mapping is vital.

Uterine cysts can be single or multiple, small or large and often compartmentalised. Large cysts may impede movement of the early conceptus, but their importance is that they are easily confused with embryonic vesicles. This cyst resembles a 16 to 17 day-old trophoblastic vesicle.

Typical features of embryonic death as detected with ultrasound include:

Failure of the embryo to appear. In this image of a day 27 pregnancy, no embryo is detectable resulting in a so-called "an-embryonic vesicle". Such pregnancies ultimately fail. Earlier in the pregnancy this vesicle was probably undersized or irregularly shaped.

Other signs are disorganisation of the placental membranes, a reduction in volume of the placental fluids and free-fluid within the uterine lumen.

Bradycardia and absence of a heart-beat are also important criteria in the diagnosis of embryonic death.

Between days 14 and 40 of pregnancy, the rate of embryonic death ranges from 10 to 15% and because pregnancy is often diagnosed at an early stage, it is important to inform owners that not all pregnancies detected with ultrasound will survive, even in apparently normal mares.

Twin pregnancy in the mare is highly undesirable and is important to identify accurately.

In this set of twins at 31 days of pregnancy, two embryonic masses can be clearly detected.

The pregnancies have fixed at the base of the same horn, termed unilateral fixation. Although they are very close together they have not developed within the same trophoblast.

If this mare had been scanned at an earlier stage of pregnancy the co-twins would almost certainly have been seen as two separate vesicles. Almost all twins in mares are dizygotic, arising from two separate ovulations.

The transducer should be gently removed at the completion of the examination, cleaned and stored carefully to avoid damage before examination of the next mare.

This article was produced by Dr. Jonathan F Pycock, B.Vet.Med., Ph.D., D.E.S.M., M.R.C.V.S. of Equine Reproductive Services who provide a wide variety of services, including:

Routine Gynaecological Monitoring of Mares
Treatment of Problem Breeding Mares
Evaluation of Stallion Fertility
Barren Mare Clinics
Artificial Insemination (Chilled and Frozen Semen)
Management of Older Breeding Mares
Behavioural Problems in Mares
For more details visit his website  Equine Reproductive Services