PREGNANCY DIAGNOSIS


Reasons

1.    It is very important to be able to tell if a mare is pregnant for management and husbandry reasons:

Pregnant mares should be managed differently from non-pregnant mares.

Pregnant mares will require further examinations to monitor the development of the pregnancy.

It is important to confirm the absence of twin pregnancies and monitor for early embryonic or foetal death Twin pregnancies are bad news in the mare and if pregnancy is diagnosed early enough, something can be done about the twin pregnancy.

 

2.   It is just as important to diagnose the non pregnant mare as soon as possible.  Some mares have prolonged luteal activity (previously termed 'persistent corpus luteum' (CL)) and the maintenance of circulating progesterone concentrations in these non-pregnant mares means they do not return to oestrus themselves. 

 

Persistent Luteal Activity in Non-Pregnant Mares

Persistence of luteal activity in the non-pregnant mare is a major cause of subfertility and is the main cause of anoestrus during the breeding season.  Traditionally, the term 'prolonged dioestrus' has been used to describe a condition where the function of the corpus luteum continues beyond its normal cyclical lifespan of 15/16 days, resulting in the maintenance of elevated circulating progesterone concentrations for longer than expected.

 

The term 'prolonged luteal activity' is now preferred.

Persistent luteal activity often arises from accessory ovulations that occur during the normal dioestrous period.  They occur in up to 20% of oestrous cycles in Thoroughbred mares (less frequently in ponies) and are not accompanied by oestrus; the cervix will remain pale in colour, dry and tightly closed.  If dioestrous ovulations occur late in the luteal phase they will be refractory (unaffected) to the effect of endogenous luteolysins, resulting in a persistent luteal phase.  These mares present great difficulty to the stud manager as they can be assumed incorrectly to be pregnant.

 

Diagnosis of mares with persistent Luteal activity

Plasma progesterone profiles are indistinguishable from those of pregnant animals.  The uterus becomes firm and tubular (tonic) and the appearance of the cervix is typical of that of pregnancy.  Transrectal ultrasound imaging fails to detect a conceptus, but a corpus luteum can be detected in the ovary.  The ability to detect the corpus luteum throughout dioestrus represents a profound diagnostic advantage of ultrasonography over rectal palpation.

 

Treatment

 

Failure of synthesis and/or release of prostaglandin F2alpha at the end of dioestrus or failure of the corpus luteum to respond to PGF2 alpha are the most likely causes of persistence of the corpus luteum.  Treatment is by the injection of a luteolytic dose of PGF2a or a synthetic analogue.  The interval between treatment and ovulation varies considerably depending upon the size of follicles at the time of treatment. 

 

Other Causes of Prolonged Dioestrous Activity

Pyometra can also be the cause of prolonged luteal activity.  Pyometra is the accumulation of substantial quantities of inflammatory secretions (usually in the form of pus) in the uterus causing its distension.  When the endometrium is severely damaged, there is extensive loss of surface epithelium, endometrial fibrosis and glandular damage causing a prolonged luteal phase, presumably due to interference with the synthesis or release of PGF2 alpha.  This is in contrast to mild endometritis with collection of small amounts of intraluminal uterine fluid, which is more likely to cause premature release of PGF2 alpha and luteolysis.

 

If diagnosed non-pregnant, appropriate measures can be taken to return the mare to oestrus and re-breed her.

 

Commercial requirements for Pregancy Diagnosis

 

1.    Later in gestation, pregnancy may have to be confirmed for sale or insurance purposes.  It can be difficult or impossible to confirm singleton (single conceptus) pregnancy at this stage.

 

2.    In many cases, the stud fee or a part of the stud fee becomes payable unless the mare is certified not in foal on the 1st October (in the Northern hemisphere).

 

Methods of Pregnancy Diagnosis

 

There are three methods used.  The first two described have only limited application and essentially the only method used routinely is a clinical examination using ultrasound.

 

1.    Absence of subsequent oestrous behaviour

 

The first is assessing the mare's behaviour by daily teasing from day 12 to 24 when pregnant mares should aggressively reject the male.  Teasing is a very important part of routine stud farm management and is an important asset to the veterinarian’s reproductive examinations.

            However, mares vary enormously in their display of signs of oestrus and to some extent this is influenced by the technique used to tease the mare.  Some mares will display oestrus signs readily, even to other mares or people.  Oestrus signs can be seen in some mares in dioestrus and late pregnancy occasionally even due to vulval irritation.  Other mares will not show oestrus signs at all.  In particular, mares that were bred for the first time (maiden mares) or mares with a foal at foot are likely candidates for failing to show oestrous behaviour.  Therefore, a significant number of mares either do not exhibit signs of oestrus, or are slow to show detectable signs using standard teasing methods despite the fact that they are in oestrus.  This is called silent oestrus. 

            The incidence of silent oestrus is thought to be approximately 6%; it is thought to have a higher incidence in maiden mares early in the breeding season and in mares with a young foal 'at foot'.  Other factors which affect oestrus behaviour include being at grass with very dominant mares, and stallion preferences.

            Mares that are in training and have been treated with anabolic steroids may be more likely to suffer from the condition due to 'androgenization'.

            Similarly, mares that are in a prolonged luteal phase will not show signs of oestrus.  The same is true for mares which undergo early embryonic death after day 15 and prolongation of the lifespan of the primary CL (pseudopregnancy type 1) or mares in which the foetus has died after establishment of the endometrial cups (approx. day 37; pseudopregnancy type 2).

 

2.   Measurement of hormone concentrations

Pregnancy diagnosis by measuring hormone concentrations in a blood (or urine) sample from the mare may be useful in certain circumstances to support a pregnancy diagnosis.  This approach may be used where their are:

1.    inadequate examination facilities,

2.    uncooperative animals,

3.    miniature horses or

4.    inexperienced veterinarian.

 

            Samples must taken at the appropriate time and results interpreted accurately.

 

a.   Plasma progesterone

Progesterone concentrations in plasma can be measured by radio-immunoassay, in which samples are sent to a laboratory and results take two or more days to obtain.  Alternatively the enzyme-linked immunosorbent assay (ELISA) tests can be used in a veterinary practice laboratory giving the results more rapidly. 

            At 17 to 22 days post-ovulation, pregnant mares should have progesterone levels above 2 ng/ml . However an incorrect diagnosis of pregnancy can occur in cases of:

1.    prolonged luteal phase,

2.    early embryonic death or

3.    mares with a short luteal phase. 

Not all mares with high progesterone concentrations are pregnant and high progesterone levels only indicate that luteal tissue is present in the ovary.  In addition, mis-timing of samples will give wrong results. 

·        Some pregnant mares may have low progesterone concentrations for periods of time. 

·        There is a marked random variation in circulating serum progesterone concentrations throughout the day.  This makes interpretation of single samples difficult if not impossible.

 

 

b.   Equine chorionic gonadotrophin (eCG)

                        This hormone is produced by fetal trophoblast cells that invade the maternal endometrium beginning around day 35 to form the endometrial cups.  There are detectable concentrations of eCG in the blood from approximately 40 days after ovulation and these concentrations usually persist at least until 80 - 120 days after ovulation when a maternal immunological reaction means thev no longer function.  The amount of eCG produced varies enormously from mare to mare.

 

False positive results in the test are seen in mares in which the pregnancy fails after the endometrial cups have formed and continue to produce eCG.

False negative results may be seen if sampling is at an incorrect time and in some mares which can be negative for eCG as early as 70 days after ovulation.

 

 

C.   Placental oestrogen determination

 

From day 65 of pregnancy onwards, serum oestrone sulphate levels are detectable and increase to peak around day 200, remaining high until after 300 days.  The oestrogens come from the foeto-placental unit especially the foetal gonads which are very large during the later stages of pregnancy.  Although small amounts of oestrogen are produced by the follicles on the mare’s ovaries, these amounts are too small to interfere with the assay. In general, this is a reliable assay and can even be performed on faeces. It is also a good indicator of foetal viability. However, its usefulness is limited by the fact that it only becomes truly reliable relatively late in pregnancy.

 

3. Clinical Examination Of Reproductive Tract By The Veterinarian

 

A clinical examination by a veterinary surgeon involving rectal palpation and ultrasound of the internal organs is the most accurate and useful method of pregnancy diagnosis. 

Nonetheless there are several potential pitfalls including the confusion of uterine cysts for conceptuses and the presence of multiple conceptuses.

 

a.    Speculum examination of the cervix

·        The vagina and cervix of the early pregnant mare are covered in pale, dry mucous membranes. 

·        The cervix is closed and resembles exactly the cervix of the mare in dioestrus.  As the pregnancy progresses, the opening of the cervix become covered in tacky, dry mucus.  Later in pregnancy (after six months) the cervix can be very relaxed.

 

b.    Rectal examination

i.      Cervix: In early pregnancy (16 to 30 days) the cervix can be palpated on the floor of the pelvis as a rigid, firm structure (as during dioestrus).

 

ii.   Ovaries: Both ovaries should be palpated although ovarian palpation contributes little to pregnancy diagnosis.  

This is because both ovaries are usually enlarged from 18 to 40 days due to follicular development and the CL is not palpable. 

From 40 to 120 days there is extensive ovarian activity with ovulations, luteinisation and development of secondary corpora lutea.

Follicular activity decreases from 120 days to term and the ovaries become small and inactive for the remainder of gestation.

The position of the ovaries up to 60 days of pregnancy is as for the non-pregnant mare.  From then on they are drawn forward and medially but remain dorsal (above) to the uterus.  From 5 months of pregnancy, the ovaries are not usually palpable.

 

iii.      Uterus: Both uterine horns and body should be palpated.  Pregnancy diagnosis is based on tone and location of uterus and contents.

 

15 to 22 days

The uterus becomes more tubular and turgid from 15 to 22 days post-ovulation and is readily palpable.  It can be difficult to feel the conceptual swelling except in maiden mares. 

 

22 to 28 days

At about 22 days the conceptual swelling develops at the uterine horn/body junction. This corresponds to fixation, when the conceptus stops migration in the uterus and becomes fixed in position, prior to implantation.  The swelling is 3 to 4 cm in diameter (about the size of a golf-ball) and it bulges ventrally.

28 to 35 days

The uterus is still turgid, and the conceptual bulge is more obvious and the embryonic vesicle is 3 to 6 cm in diameter.  The uterine wall over the conceptus begins to feel thin.

 

35 to 60 days

As the conceptus grows the swelling becomes larger and spherical and appears more as a fluid sac.  By 42 days it is about 5 x 7 cm (the size of a tennis ball).  The uterine tone around the bulge begins to decrease so that the swelling becomes less tense.

 

 

60 to 90 days

By 60 days the swelling is about 12 cm in diameter and fills the pregnant horn.  After 60 days the pregnancy is like an elongated football and starts to involve the uterine body.  One must be careful not to confuse it with the bladder or a case of pyometra.  The uterus migrates cranially.

Between days 60 and 100, the uterus is low within the abdomen and the foetus can not usually be palpated;

From 4 to 5 months onwards the foetus can usually be palpated.

C.   Ultrasound Examination

 

Diagnostic ultrasound plays a pivotal role in the reproductive management of the mare and no deleterious effects of ultrasonography have been reported in man nor in the equine. 

A thorough understanding of normal ultrasonographic anatomy is vital for veterinarians involved in broodmare work.

 

Principles of diagnostic ultrasound

      Diagnostic ultrasound utilises sound frequencies between 2 and 10 MHz.

      Ultrasound is produced by application of an alternating voltage to piezoelectric crystals which change in size and produce a pressure or ultrasound wave.  Returning echoes deform the same crystals which generate a surface voltage.

• Most diagnostic ultrasound machines use the principle of brightness modulation (B-mode) where the returning echoes are displayed as dots, the brightness of which is proportional to their amplitude.

• Real-time B-mode ultrasound is a dynamic imaging system where information is continually updated and displayed on a monitor.

• Ultrasound is attenuated within tissues and attenuation is related to the density of the tissue, the heterogeneity of the tissue and the number and type of echo interfaces.

• Bright (specular) echoes are produced when a large proportion of the beam is reflected back to the transducer; these echoes are displayed as white areas on the ultrasound machine screen.

• No echoes are produced when the sound is transmitted and not reflected; these areas are displayed as black on the ultrasound machine screen.

Ultrasound terminology

      Tissues that markedly reflect sound (such as bone and metal) appear white on the ultrasound screen and are called echogenic.

      Tissues that transmit sound (such as fluid) appear black on the ultrasound screen and are called anechogenic (or anechoic).

           Tissues that allow some transmission and some reflection (such as most soft tissues) appear as varying shades of grey and are called hypoechogenic or hyperechogenic depending upon their exact appearance. For example, ovaries usually contain follicles (which are anechoic), and may contain luteal structures (which are relatively echogenic - varying shades of greywhite).

 

Applications

Ultrasound now has numerous applications in equine practice.

·        Ultrasound imaging of tendons has become a standard practice in assessing damage to and the repair process in flexor tendon damage and can also b used in the imaging of the suspensory apparatus and the limb joints.

·        Ultrasound is also useful in assessing structural and functional aspects of the equine heart (echocardiography) and is also finding increasing use in the diagnosis of various thoracic and abdominal conditions.

·        In the area of reproduction, ultrasound is used increasingly in both the stallion and mare in the following areas:

Stallion:

o       Assessment of scrotal contents and penile disease;

o       Assessment of accessory sex organ health.

Mare:

·        Staging the oestrous cycle by revealing ovarian and uterine structure.

2. Identifying uterine problems such as endometritis, fluid in the uterus, endometrial cysts or failure of involution (post-foaling).

o       Pregnancy diagnosis

o       Identification of twin pregnancies.

o       Gender Determination

o       Assessment of Foetal Well-Being.

 

Restraint

To carry out ultrasound 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.  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. 

 

Diagnosis of early pregnancy using ultrasound: Day 10-11

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

            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 twin arose from a later ovulation.

This fact, together with the mobility and relatively small size of the conceptus make 14 to 15 days the optimal stage of pregnancy to diagnose twins and crush one co-twin. We will discuss diagnosis of twins in upcoming lectures.

 

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.

 

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 22 of pregnancy the conceptus is irregular in outline.  This irregular shape is normal and is not an indication of imminent pregnancy loss.  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.

 

All embryos should be detectable by day 24. 

 

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.

 

            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 28-29 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.

Note the enlargement of the allanioic 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 often 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.

 

By day 40 of the 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.

 

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.

 

Ovarian Examination

Examination of the ovaries is important at every examination for pregnancy to provide information on the number and appearance of the CLs.  Mares with twins almost always have two corpora lutea. A CL readily detectable on ultrasound is usually producing progesterone.  Ultrasonographic assessment of the CL may be useful in assisting a decision to provide exogenous progesterone or progestogen.  During the examination of pregnant mares between 40 and approximately 150 days, large follicles and supplementary CLs are frequently found in addition to the primary CL.  The supplementary CLs are often as large as 6 to 8 centimetres, with thick echoic walls and anechoic centres crisscrossed by echoic trabeculae.

 

 

Timing of Routine Pregnancy Scans

·          The first examination should be around 15 days after covering to diagnose pregnancy early and treat twins. 

·          A second exam around 26 to 30 days after covering allows the vet to check for normal development of the pregnancy and confirm that there are no twins.  These timings are arbitrary.

·          Extra scans may be needed in mares which are prone to twinning, have many cysts or have lost pregnancies before.  The cost is an important consideration as with many aspects of horse breeding, a compromise must be struck between what is best and what is practical in economic terms.

·          If a mare is only able to be scanned once on economic grounds then this should be around day 28 when an obvious embryo with heart beat should be visible. 

·          Mares scanned in foal in the breeding season should all be checked in autumn by internal examination to make sure that they are still pregnant.

 

 

Pregnancy Diagnosis: Protocol (When Ovulation Time is Known)

 

First exam                         Day 14 to 15

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 confirmed 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.

Some mares, for example mares with several uterine cysts, may require additional examinations.

 

Multiple ovulation

            Double ovulations occur during 8-25% 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 highly undesirable. 

Rectal palpation alone can be misleading in detecting a double ovulation, particularly when the two follicles are on the same ovary.  The use of ultrasound examination of the ovaries, which should routinely be performed in conjunction with a thorough rectal examination, usually allows detection of a double ovulation.  Sometime the ovulatory area can appear indistinct for the first 24 hours, and in these cases the mare should be checked again the two days later when it can be more easily seen if there are two CL’s.  It is 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. The advent of ultrasound imaging has provided a method of more readily managing twin pregnancy in the mare.  Consequently, multiple ovulations in the mare should not be regarded as a reason for withholding mating.  In fact, pregnancy rates are improved after twin ovulation.

 

Foetal Gender Determination

            Knowledge of the gender of an unborn foal is beneficial for both research and commercial purposes.  Commercially, the value of a mare may be substantially increased if she carries a foal of the desired gender.  Numerous techniques have been developed to determine embryonic or foetal gender in humans and domestic species and some of these techniques have been applied to horses.  Separation of X-chromosome and Y-chromosome sperm before fertilisation has been attempted with some success.  Detection of the male specific H-Y antigen on the equine blastocyst has been demonstrated and the embryonic gender confirmed with karyotypes (shromosomal analysis).

Ultrasonography offers a non-invasive technique to determine the viability and gender of a foetus and has been used for this purpose in humans, cattle, and horses. In humans, gender is diagnosed by identification of foetal external genitalia after day 140 of gestation with accuracy nearing 100%.  In cattle, foetal gender determination has been demonstrated by ultrasonic imaging of either the scrotum or the mammary glands or by identifying and locating the genital tubercle.  In horses, foetal gender can be accurately determined by identifying and locating the genital tubercle.

            The genital tubercle is the embryonic forerunner of the penis and clitoris.  It can be identified by gross inspection of removed equine foetuses as small as 10 to 12 mm (approximately postovulation day 28).  When first identified, the genital tubercle is located on the mid-line between the rear legs, and gender cannot be determined by gross inspection of the embryo.  Foetal gender of removed specimens can be determined after days 40 to 45.  At this time, the clitoris is located along the posterior border of the perineal region in the female foetus; the penis is located in the anterior inguinal area in the male foetus.  In the male, the prepuce is first noted by day 77 and becomes pendulous by days 115 to 120.  The scrotum is small and empty in the day-80 foetus. 

The genital tubercle cannot be identified ultrasonically on day 35 or 36.  After this time, the genital tubercle is identified with increasing frequency through day 54, at which time it can be identified in all mares.  Before day 48, the genital tubercle is located on the mid-line between the rear legs.  After day 48, the location of the genital tubercle relative to surrounding structures begins to change; it moved toward the umbilicus in male foetuses and toward the tail in female foetuses. 

As foetal age increases, certainty scores gradually increase, reaching a maximum certainty on day 60.  After day 70, gender can not always be determined.  This is attributed to difficulty in obtaining an adequate view in the older foetuses.  The optimal time for gender diagnosis was from days 59 to 68, at which time the genital tubercle is readily accessible and assignable to a location characteristic of its gender with a high level of certainty.

Gender determination after this time point is more difficult due to the position and accessability of the foetus.

 

FOETAL ULTRASONOGRAPHY

The advent of diagnostic ultrasonography in the early 1980s has revolutionized the routine gynecological management of the mare.  Transrectal ultrasonography is used routinely, often repeatedly, during the first weeks of pregnancy to identify and monitor the development of the early equine conceptus and subsequent foetus.  This is in contrast to the current obstetric care regimen for pregnant women who normally have two midgestational scans, the first scan at approximately 16 weeks of pregnancy (essentially to confirm gestational age) and the second scan at about 22 weeks to detect any developmental abnormalities and confirm continued satisfactory growth.  Further scans are not made unless there is a complication with the pregnancy.  This difference, to a certain extent, reflects the differing priorities between the management of the pregnant mare and the pregnant mother and also the relative ease of diagnosis.  In the latter, determining "dates" and identifying developmental abnormalities are the major priorities.  In addition, in human obstetrics prepartum detection of foetal distress and disease has become an essential aspect of the management of the problem pregnancy.  In these cases ultrasonography has proved invaluable, particularly where placental bleeding is present or where invasive procedures such as amniocentesis, chorionic villus sampling, and foetal blood or tissue sampling is necessary. 

With the improvement in the clinical management of the sick, newborn foal during the last 10 years, the need to develop techniques for assessing the status of the equine foetus in late term has become apparent.  Hopefully, by detecting the distressed foetus and by diagnosing disease and prematurity, the management of the sick neonate will be further improved.  To achieve this end a number of research workers have applied diagnostic ultrasonography to the assessment of the late gestational equine foetus.

The indications for foetal ultrasonography in early to mid gestation include assessment of foetal viability and foetal sexing, which is best performed at about 60 days of gestation.  Indications for foetal ultrasonography in mid to late pregnancy in the mare include vaginal discharge, premature mammary development and lactation, systemic maternal disease (colic, trauma, surgery), and a history of previous problems in late gestation such as abortion, stillbirth, prematurity, and neonatal septicemia or death.

 

Foetal Imaging Techniques

Foetal imaging can be performed either from a transrectal or transabdominal approach.  Both means of imaging the foetus have their own inherent limitations and advantages.  Transrectal imaging only allows effective imaging of the foetus during the first 60 to 80 days of gestation; however, it is the preferred route for examining the foetus to determine foetal gender (60 to 70 days) and to measure the dimensions of the foetal eye orbit at more than 120 days.  Most foetal ultrasonography is performed transabdominally because it allows the maximum visualization of the foetus and placenta.  The foetus, often from as early as 60 days of gestation, can be visualized through the mare's ventral abdominal wall due to the close proximity the uterus adopts to the ventral abdomen. 

          A specialised form of ultrasound transducer called the linear array transducer is used. This has the advantage of giving an image that is easier to interpret, particularly as the foetus increases in size during the later stages of pregnancy.  Good preparation of the mare is important to ensure high-quality images and to reduce artifacts.  Ideally, the mare should be restrained in stocks and the skin closely clipped.  The mare should not be sedated as this will affect foetal activity and heart rate The transducer is then moved systematically from side to side and up and down until the foetus is located.  Usually, the relatively anechoic foetal fluids are most obvious along with the foetal thorax and contracting heart. It may be possible to image the foetal head where the anechoic eyeball and orbit are the most prominent features.  The foetal neck with tracheal rings and pulsatile carotid arteries can be recognized before joining the foetal thorax.

 

FOETAL MEASUREMENTS

In pregnant women a number of different foetal measurements are commonly made.  Crown-rump length is used in early gestation and is accurate prior to 13 weeks of gestation.  Later in gestation the femur length, and abdominal circumference are measured and used to determine both foetal size and weight.  The determined values are then compared, using growth charts, with the expected values for that age of foetus.  A number of different growth charts are available to obstetricians to help them identify the growth-retarded foetus.  Unfortunately, in the equine foetus its large size in the latter stages of pregnancy restricts the use of similar measurements.  However, two structures that have been used, which are relatively small in size and can be measured sequentially, are the dimensions of the foetal eye orbit and the diameter of the foetal aorta. Unfortunately, neither of these techniques provides particularly reliable data on foetal size and growth.

            A more reliable method of assessing foetal well-being may be forthcoming in the form of a Foetal Biophysical Profile. This is a composite method to assess foetal well being by determining if the foetus shows signs of foetal asphyxia (shortage of oxygen). The parameters used are:

Foetal breathing movements

Gross body movements

Foetal body tone

Foetal Heart Rate

Amniotic Fluid Volume.

 

These 5 parameters are assessed  and given a score (2 is normal; 1 is questionable and 0 is abnormal). A score of 8-10 is normal and a score below 8 or 10 is abnormal. This method is a good predictor of foetal asphyxia and in a recent trial did provide good advance warning of abnormal or dysmature foals. In such cases, the mare might be induced and intensive care could be available for the foal at birth. Intra-uterine treatment of foals is not an option as yet.