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Reproductive
Ultrasonography The Mare INTRODUCTION Few
people predicted the impact that ultrasonography would have on equine
reproductive management and understanding of reproductive physiology. The
ability to examine a mare's reproductive tract non-invasively with
ultrasonography provided the opportunity to diagnose pregnancy earlier than by
rectal palpation, effectively manage twins and detect impending early embryonic
death (EED). However, ultrasonography should not be limited to these areas. It
can be used to diagnose uterine pathology, such as intrauterine fluid, air,
debris, cysts and occasionally abscessation and neoplasia. In addition,
ultrasonographic examination of the
ovaries may aid in determining stage of oestrous cycle, status of preovulatory
follicles, development and morphologic assessment of the corpus luteum (CL) and
in interpreting ovarian irregularities, such as anovulatory or haemorrhagic
follicles, neoplasia and peri-ovarian cysts. The costs of equipment initially
resulted in a rather limited application of reproductive ultrasonography.
Clients enthusiastically support use of ultrasonography to detect pregnancy.
However, the same fee schedules for routine examination before and/or after
breeding are not as easily accepted. An approach that allows us to scan multiply
while still keeping clients and farm managers happy is something all of us
strive to organise each year. A more logical and thus practical approach to
diagnosis and treatment of physiological and anatomical abnormalities of the
mare's reproductive tract would be forthcoming if we can continue to develop a
means to use the equipment more routinely. In
addition, valuable information would be available from correlation of fertility
data with normal and abnormal ultrasonographic observations. Regardless,
informed clientele prefer routine ultrasonography and its use results in a more
interactive approach to farm management with an increased awareness of the
events associated with breeding, ovulation and early foetal development. If
there is a drawback with its use, then most commonly it is manifest as some
clients wishing to purchase their own equipment and pursue their own diagnoses. The Uterus Pregnancy Fertilisation
occurs at the ampullary/isthmus junction of the oviduct, and requires a viable
oocyte and spermatozoon. The first maternal recognition of pregnancy may be as
early as 48 hr post-ovulation in mares. This
is associated with production of an immuno-suppressive agent, a
pregnancy-specific protein called early pregnancy factor (EPF).
Early pregnancy factor has been detected in mice, sheep, humans and mares
and may have promise for future early detection of pregnancy and early embryonic
death. Another event in maternal recognition of pregnancy occurs on or before 6
days post-ovulation. Fertilised ova
are transported from the oviduct through the utero-tubule junction and into the
uterus by 5 to 6 days post-ovulation, while unfertilised ova (UFO) are generally
retained in the oviduct. After fertilisation (day 0) and initial cleavage, each
equine blastomere (cells produced by cleavage) divides approximately every 24
hours. Based on oviductal flushes,
it is common to collect 4 to 8-cell embryos on day 2 post-ovulation and 8 to
16-cell embryos on day 3 (McKinnon & Squires. 1988). A 32- to 64-cell embryo
is classified as a morula and is the youngest developmental stage of embryo that
can be harvested from the uterus. Generally, 6-day embryos are late morulas or
early blastocysts. A blastocyst is recognised by development of a blastocoele
cavity within the embryo. The blastocoele cavity, or yolk sac, is fluid-filled,
and continued expansion of the blastocoele allows ultrasonographic determination
of pregnancy as early as 10 days post-ovulation. Day-7 embryos are generally
expanded blastocysts. Embryos are generally visible to the naked eye by 7 to 8
days post-ovulation. A third event in maternal recognition of pregnancy occurs
between 12 and 16 days. During this time the conceptus is extremely mobile and
it is postulated that the conceptus prevents release or inhibits function of
prostaglandin F2-alpha that would normally destroy the CL resulting in a return
to oestrus(Hershman & Douglas. 1979; Nilsfors, Kvart, Kallings, Carlsten,
& Bondesson. 1988). The conceptus may also produce substances that are
luteotropic. For diagnosis of
pregnancy at 10 to 12 days, a 5 or 7.5 MHz transducer is necessary. However, due
to embryonic loss in early pregnancy, it is inappropriate to discontinue the
teasing program after initial examination for pregnancy. From a practical
standpoint, the first examination could be postponed until approximately 18 to
20 days post-ovulation, thus eliminating scanning of mares that are destined to
return to oestrus. One exception would be scanning of breeds that have a history
of twinning or multiple ovulations (ie. Thoroughbreds). These mares must be
examined at day 12 to 15 post-ovulation to most effectively manage manual
embryonic reduction. Frequency of
subsequent scans will depend on such factors as presence of twins, size and
quality of the vesicle and embryo, availability of the mare and economics.
Timing of the initial pregnancy examination depends on factors such as breed,
economics, convenience and client education. Characteristics
of the Conceptus: Days
10 to 17 Ultrasonographic
scanning has resulted in an increase in our knowledge of dynamics of early
pregnancy. Ultrasonographic images of the conceptus at various stages have been
grouped together for convenience. It has been shown that the early equine
conceptus is highly mobile within uterine lumen. Regardless of the side of entry
into the uterus, the equine conceptus moves between the uterine horns and
uterine body. Small vesicles (day 10) are spherical and found most frequently in
the uterine body. Trans-uterine movement occurs at intervals of less than 2 to 4
hr (Ginther. 1983). Mobility begins to decrease by day 15, and after day 17
trans-uterine migration can no longer be detected (Ginther. 1983; Ginther.
1983). Thereafter, the vesicle is fixed at the caudal portion of one of the
uterine horns. Extensive mobility of the early conceptus may be due to the
spherical form and turgidity of the vesicle, and longitudinal arrangements of
endometrial folds. Researchers have demonstrated that restriction of embryonal
movement resulted in pregnancy failure (McDowell, Sharp, Grubaugh, Thatcher,
& Wilcox. 1988). These investigators suggested that pregnancy failure was
caused by inability of the conceptus to reduce uterine secretion of
prostaglandin F2µ.
When scanning for an early vesicle, the transducer should be moved slowly so the
image or tissue slice visualised (2 to 3 mm wide) is not passed over the vesicle
too rapidly. A systematic technique should be developed to avoid omitting or
scanning too rapidly a portion of the reproductive tract.
Because the vesicle is moving, it may be found anywhere within the
uterine lumen from the tip of a uterine horn to the cranial aspect of the
cervix. It should be emphasised that early detection of an embryonic vesicle
requires a high frequency transducer (5 MHz) and a high-quality screen.
Frequently, ultrasonographic images of a 10 to 14 day vesicle has a bright
echogenic line(specular reflection) on the dorsal and ventral poles with respect
to the transducer. These are not associated with the embryonic disk or other
structures of the developing conceptus. A water-filled balloon (1.5 cm diameter)
placed in the uterus will have similar, if not identical, ultrasonographic
characteristics. Days
17 to 22 The
vesicle is spherical in shape before day 17. The vesicle grows quickly between
days 11 and 17 and almost doubles in size every day (McKinnon, Squires, &
Voss. 1987; McKinnon, Squires, & Voss. 1987). The vesicle has a growth
plateau between days 17 to 26, then growth resumes at a slightly slower rate.
After day 17, the vesicle is often irregular in shape. Fixation of the early
conceptus on days 16 to 17 apparently is due to increased uterine tone and
thickening of the uterine wall as well as rapid growth of the conceptus
(Ginther. 1983). Increasing uterine tone may explain why the vesicle changes
shape as pregnancy advances. Fixation generally occurs in the caudal portion of
the uterine horn near the bifurcation (corpus cornual junction). In post-partum
mares, the previously gravid horn provides less restriction, and thus the
conceptus generally fixes in the opposite uterine horn.
Fixation occurs with greater frequency in the right horn in maiden and
barren mares (Ginther. 1983). Orientation is defined as rotation of the
embryonic vesicle so the embryo proper is on the ventral aspect of the yolk sac
(Ginther. 1983). On day 14, the vesicle is highly mobile and the embryo is
probably not orientated. Shortly after the end of the mobility phase (days 15 to
17), the dorsal uterine wall begins to enlarge and encroach upon the yolk sac.
Encroachment is enhanced by increasing uterine tone. The disproportionate
thickening and encroachment of the uterine wall on the vesicle, in addition to
the massaging action of uterine contractions, cause the vesicle to rotate so the
thickest portion of the yolk sac (embryonic pole) assumes a ventral position
(Ginther. 1983). Hypertrophy of the uterine wall is especially prominent on each
side of the dorsal midline. This probably accounts for the midline location of
the apex of the triangular-shaped vesicle, and the thinness of the uterine wall
ventrally. The embryo is first detected ultrasonographically within the vesicle
at day 20 to 25, and is most commonly observed in the ventral position. The
heartbeat is commonly detected about day 22, and is an important indicator of
the embryo's well-being. Days
22 to 55 It
is important that the ultrasonographer understands and interprets clinically the
growth of the allantois, which is initially recognised on around day 24, and
concurrent with its expansion, the contraction of the yolk sac. The interplay of
growth between these two fluid-filled structures result in the embryo moving
from the ventral (day 22) to dorsal (day 40;) aspect of the vesicle. After day
40, the yolk sac degenerates, and the umbilical cord elongates from the dorsal
pole, permitting the foetus to gravitate to the ventral floor where it is seen
in dorsal recumbency from day 50 onward. Apposition of yolk sac and allantois
results in an ultrasonographically visible line normally orientated
horizontally. On occasion, we have identified this junction in a vertical
configuration, and believe it has no deleterious effect on continuing pregnancy.
However, there is one report that suggests that these mares have a lower than
commonly accepted normal progesterone levels, which may be associated with
failure to orientate properly(Griffin, Carnevale, & Ginther. 1993). Twin
vesicle walls, when in contact, generally appear as an ultrasonographically
visible, vertically oriented line. With knowledge of the approximate stage of
gestation and growth characteristics of the conceptus, the clinician can
differentiate between presence of an abnormally orientated singleton or of two
apposed yolk sacs (twins). Developmental abnormalities are more easily detected
with ultrasonography than rectal palpation. On one occasion, we used
ultrasonography to detect an abnormally developing foetal monster, with
excessive fluid in the cranium. Obviously,
with the appropriate equipment, accurate aging of the young foetus is possible
by ultrasonography. However, no reliable method for accurately determining
foetal age, late in gestation, has been developed. Foetal eye size determined by
ultrasonography, has been correlated (r = 0.92) with foetal age (McKinnon, Voss,
Squires, & Carnevale. 1993). Measurement of foetal eye size was made with a
5 MHz transducer in mares of known gestational age. Eye size was calculated from
the sum of width plus length. Identification of the eye was not difficult due to
dorso-pubic positioning of the foetus after day 90. Early
studies on the efficacy of ultrasonography for pregnancy diagnosis have
demonstrated extreme accuracy after day 15 (> 97.4%) (Selway. 1983; Simpson,
Greenwood, Ricketts, Rossdale, Sanderson, & Allen. 1982).False positive
diagnoses were related to misinterpretation of uterine cysts and false negatives
were attributed to operator inexperience and scanning too rapidly. These studies
were performed with 3 MHz transducers and our abilities to diagnose pregnancy
have improved further with the advent of 5 MHz transducers, better image
quality, more information on early conceptal development and increased operator
experience. This experience has taught us that the following factors are
important in accurate identification of an early pregnancy: 1) Equipment quality
and transducer frequency: 2) Mare restraint and examination environment: 3) Age
of the conceptus at the time of examination and interval between multiple
ovulations: 4) Operator experience: and 5) Sexing
the foetus Accurate
knowledge of the gender of an unborn foetus gives the owner an opportunity to
decide to sell or keep a pregnant mare. If performed early enough it may allow
the mare to be aborted and rebred in the same breeding season. The technique of
sexing, using ultrasonography, relies on visualisation of the genital tubercule
and its location relative to the tail or the umbilicus. The genital tubercule
becomes the penis in the male and the clitoris in the female. The genital
tubercule is distinguished close to the umbilicus in the male and the tail in
the female. From post mortem studies on accurately aged foetuses it was not
possible to see genital tubercule migration until after 40-45 days (Bergin,
Gier, Frey, & Marion. 1967). “By 45
days, gross external sex determination is possible. The clitoris in the female
fetus is located along the posterior border of the perineal region and extends
in a posterior direction on a horizontal plane. The penis of the male fetus is
located in the anterior inguinal area and extends in an anteroventral
direction” (Bergin, Gier, Frey, & Marion. 1967). Sexing the foetus
with ultrasonography has been shown to be accurate under both research and farm
conditions (Curran & Ginther. 1989; Curran & Ginther. 1991).Under
research conditions it was first established that the most accurate time for
diagnosis was between day 59-68 (Curran & Ginther. 1989).In this instance 92
of 92 female diagnoses were correct and 138 of 143 (97%) of male diagnoses.
Under farm conditions 85 mares with pregnancies between 50 and 99 days were
evaluated (Curran & Ginther. 1991).Foetal gender was able to diagnosed in 75
of 85 mares (88%) and was accurate in 73 of the 75 cases (97%). The average time
for location and identification of position of the genital tubercule was only 1
min and 17 seconds. The authors conclusions were that foetal gender diagnosis is
limited in early pregnancy due to undifferentiated foetuses and in later
pregnancy due to inability to adequately view the foetus. However, in the older
foetus, when a diagnosis was made it was accurate. A further study demonstrated
that using a 3.5 MHz probe it was possible to extend the time of diagnosis up to
6 months, however only 90% of examinations were completed (Curran & Ginther.
1993). Because
the time period of sex determination is coincident with the invasion and
maturation of the foetal trophoblast cells to the uterus (endometrial cups),
abortion and rebreeding are not an option. This has led to a slow uptake in the
number of clients that have requested this service from our practice. In most
instances the animals have been for sale. Twins Twins
are important for a variety of reasons. Firstly, historically twins have been
the single most important cause of abortion in Thoroughbreds (Acland. 1987;
Acland. 1993) and secondly, regardless of the breed twinning is a huge cause of
reproductive wastage as most pregnancies terminate in early foetal resorption or
loss, late term abortions, or the birth of small growth retarded foals. Mares
aborting twins in late gestation frequently have foaling difficulties, damage
their reproductive tracts and are difficult to rebreed, presumably due to
delayed involution of an oversized uterus. Foals born alive are frequently small
and demonstrate intra-uterine growth retardation. Their survival rate is low and
for many, long term survival necessitates expensive sophisticated critical care.
For all of the above reasons twins are a disaster and should be avoided at all
costs. An important philosophy that owners, stud, managers and veterinarians are
all coming to grasp with is that twins are preventable. Twins
are linked with breed, season, nutrition and a familial predisposition. More
twins occur in Thoroughbreds, Draughthorses and Warm-bloods compared to
Standardbred and Ponies. In early pregnancies diagnosed with ultrasonography,
Thoroughbreds had an incidence of 97 of 629 pregnancies (15.4%) and
Standardbreds an incidence of 39/634 (6.1%) (Bowman. 1986). The number of
abortions due to twins in one study was lowest in cold-blooded horses and in
Arabian horses (Byszewski & Gromnicka. 1994). In an other study multiple
ovulation rates were reduced in foaling mares (lactating) compared to barren
mares and maidens and more frequent in Thoroughbreds (19%) compared to Quarter
horses (9%) and Appalosa’s (8%). Multiple ovulation rate is directly related
to twinning and together with twining was demonstrated as highly repeatable
within mares. This study also showed that withholding breeding did not prevent
twins (Ginther, Douglas, & Lawrence. 1982). Twinning was the cause of
6.1% of equine abortion and
still birth in central Because
the expected outcome for mares with twins is so poor for either the mare or the
resultant foal(s) it is our responsibility to successfully manage early
pregnancies such that no mare delivers or aborts twin foals. In consultation
with farm managers, owners and clients we must to utilise available equipment
and technology commensurate with economic constraints and other owner/manager
preferences to diagnose twin pregnancies as early as practically possible. It is
the responsibility of a veterinary profession to adequately inform
owners/managers/clients of reasons why twins may not be diagnosed. In
the future it is possible that veterinarians will be held accountable for
failure to diagnose twins, especially in circumstances where owners expect
sophisticated reproductive services and mares are examined repeatedly. Reasons
why twins may be missed despite repeated examination are: 1) difficulty
distinguishing structures. This may be related to a poor scanning environment ie,
to much light, or poor display characteristics of the ultrasound, or mare
movement and/or lack of restraint. 2) variable growth patterns. 3) inability to
detect heart beats of adjacent embryos. 4) operator experience and 5) resolution
of the equipment. In our experience the most common cause to misdiagnose early
twin pregnancy is scanning prior to the recognition of a second (asynchronous
ovulation) pregnancy. Another reasonably common occurrence would be scanning too
quickly. Twin
pregnancy has been a common cause of abortion (Jeffcott & Whitwell. 1973).
The incidence of abortion is decreasing (Hong, Donahue, Giles, et al. 1993) and
in the German Thoroughbred industry has declined from 2.7% to 1.7% since the
event of ultrasonography (Merkt
& Jochle. 1993) Origin of twins Many
non equine veterinarians would be surprised to learn that twins in the horse are
almost, if not exclusively associated with a double ovulation. Until recently
only one reference alluded to the identification of an identical twin (Rooney.
1970). In this text Rooney states “I
have only seen one definite example of identical twinning (of some 600 such
abortion examined); there were two amniotic sacs and a single allantochorion”.
Recently an article appeared that demonstrated that identical triplets had
occurred (confirmed with DNA analysis) which were first suspected after video
endoscopy showed they were all in one chorionic sac (monochorionic) (Meadows,
Binns, Newcombe, Thompson, & Rossdale. 1995). It was disappointing to note
that there was no mention by the authors of the number of CL’s detected by
ultrasonography. Reasons for lack of identical twins in the horse would appear
to be related to the equine capsule. The capsule forms in embryos aged around 6
days, shortly after their entry into the uterus (McKinnon, Carnevale, Squires,
Carney, & Seidel, Jr. 1989). When embryos were cultured prior to the
formation of the capsule hatching occurred similar to as occurs in the bovine
(McKinnon, Carnevale, Squires, Carney, & Seidel, Jr. 1989), however when
embryos were cultured after formation of the capsule the zona pellucida
continued to become progressively thinner and finally fell away from the
developing conceptus. The postulated mechanism for identical twin formation in
other species is embryo bisection associated with hatching and pinching of an
early embryo (A Trounson personal communication - quoted in (McKinnon, Voss,
Squires, & Carnevale. 1993)). As an interesting aside 70 of 111 (63%) of
aborted twins were the same sex which indicated to those authors (Merkt,
Jungnickel, & Klug. 1982) that perhaps splitting of the embryo occurs. This
observation may be by chance alone. Another interpretation would be an improved
efficiency of eliminating opposite sexed foetuses (earlier in gestation). A more
recent study (Prohl, Busch, & Schutzler. 1994), recorded that of 35 twin
pairs, 65.7% were of opposite sexes. This study is in contrast to the previous
one and serves to highlight dangers inherent in extrapolation of anything but
large sets of data. The recent publication of identical horse triplets (Meadows,
Binns, Newcombe, Thompson, & Rossdale. 1995) stated they did not arise from
separation of blastomeres during early cleavage stages, as division at this time
would have given rise to individual sets of extraembryonic membranes.
“Separation of the inner cell mass must have occurred prior to gastrulation in
order to result in each embryo sharing the outermost chorion while at the same
time having an individual amnion” (Meadows, Binns, Newcombe, Thompson,
& Rossdale. 1995). Originally
twins were hypothesised to have occurred more frequently from asynchronous
ovulations (Ginther. 1983). This author found double ovulation and twins were
seen more frequently in barren mares (11% and 6% respectively) compared with
lactating mares (5% and 1% respectively) and that 9 twin pregnancies from 32
mares were associated with ovulations two days apart (asynchronous) compared to
0 out of 19 for synchronous ovulations. Many of these original observations were
made prior to ultrasonography and as a good example of these inherent diagnostic
difficulties the same author quoted 70% of twins arriving from one detected
ovulation (based on analysis of multiple veterinarians breeding farm records)
(Ginther. 1982; Ginther. 1983). Subsequently it was shown that twins were as
likely to result from synchronous versus asynchronous ovulation (Ginther. 1987)
and that pregnancy rate per follicle was identical for double ovulations on
opposite ovaries to that obtained from single ovulations per cycle but was
higher than the pregnancy rate per follicle when double ovulations occurred on
the same ovary. These results indicated no embryo reduction prior to first
diagnosis of pregnancy with ultrasonography in bilateral ovulators as each ovum
had the same chance of developing into a day 11 conceptus as an ovum from single
ovulators. In unilateral double ovulators the lower day 11 pregnancy rate per
ovulation compared with bilateral ovulators and single ovulators was
attributable to a greater frequency of mares with no embryonic vesicles rather
than to a greater frequency of mares with one vesicle (Ginther & Bergfelt.
1988). Outcome of twin pregnancies 1) Non intervention Prefixation Days 11-16 Embryo
reduction before or on the day of fixation was not considered an important
aspect of the natural correction of twins (Ginther. 1989). Diameter and growth
rates on days 11-16 were similar between singleton and twins and the presence of
two vesicles did not have a direct effect on diameter other than that
attributable to their age. The probability of a mare loosing one or both
vesicles of a set of twins from identification prior to fixation is minimal and
approximates that of early embryonic
death for the same time period (per vesicle). The recognition of twin
pregnancies prior to fixation day (day 16) is dependant on the day of
examination relative to the day of ovulation.
Asynchronous ovulations occasionally result in a gross disparity in vesicle
size, sometimes as much as 5 days ie. identification of a day 11 and a day 16
vesicle concurrently. In instances such as this, examination one day earlier
would have failed to detect the younger of the two pregnancies. Recognition that
all twin pregnancies occur from multiple ovulation dictates mandatory
re-examination of all mares that have two CL’s and only a
single vesicle detected prior to fixation (day 16 ). Recognition prior to
fixation is also dependent on operator experience, resolution of the equipment
(5 MHZ preferred), monitor capabilities, restraint and other facilities (ability
to darken the environment), the presence of uterine cysts and the skill of the
examiner. Recognition post fixation The
recognition of unilaterally fixed twins from day 17 through to 21 (prior to
clear recognition of the developing foetus within the vesicle) is probably the
most difficult time to determine if there are twins present.
Ultrasonographically all that is present is a thin line (the apposition of the
two yolk sacks) running approximately in the middle of a slightly over-sized
vesicle. Recognition of the foetus(s)
within the vesicle a few days later makes differentiation easier. Occasionally
an inexperienced operator may confuse an abnormally orientated 28 to 30 day
single pregnancy with 17 to 20 day unilaterally fixed twins. From days 22 to 60
the presence of multiple foetuses, umbilical cords and general excess in the
number of visible membranes should alert the practitioner to the likelihood of
more than one pregnancy. The junction between two developing foetuses (after 30
days) between the two allantochorions results in a common membrane from the area
of apposition. This common membrane has been referred to recently as the twin
membrane (Ginther & Griffin. 1994) and has diagnostic potential,
particularly late in pregnancy when it might not be possible to view both
foetuses transrectally (>100 days). After 100 days, careful transabdominal
ultrasonography may be necessary to determine the presence of twins. Postfixation Days 17 to 40 The
outcome of foetuses post fixation is dependent upon the nature of their
fixation. Unilateral (both fixed together at the same corpus cornual junction)
fixation reduction is much higher than bilateral (one on each side) fixation
reduction. Fortunately, unilateral fixation is much higher (approximately 70%)
compared to bilateral (30%). In one study of 31 mares with twin embryonic
vesicles, unilateral fixation (71%) was more frequent (p< 0.05) than was
bilateral fixation (29%) (Ginther. 1989). In 28 mares with known ovulatory
patterns, synchronous ovulations did not affect the type of fixation (9/17
unilateral , 8/17 bilateral). However for asynchronous ovulators the frequency
of unilateral fixation (10/11) was greater (p< 0.01) than the frequency of
bilateral fixation (1/11). The incidence of embryo reduction was greater (p<
0.01) for unilateral fixation (14/19) than for bilateral fixation (0/9) and was
greater (p< 0.05) for asynchronous ovulators (9/11) than for synchronous
ovulators (5/17) (Ginther. 1989). When reduction occurs with unilateral
fixation, it is most commonly effected early (prior to recognition of the
foetus). In one study, 10 of 14 reductions occurred prior to detection of either
embryo . The degree of synchrony of ovulation
also affected reduction. Early reduction occurred in 8 of 11 mares with
asynchronous ovulation and for 17 synchronous ovulators none reduced early, 5
reduced late and 12 didn’t reduce at all (Ginther. 1989). In another study the
incidence of reduction was higher
for unilateral fixation (41/48) (85%). In cases of unilateral fixation, 22 of 22
mares with vesicles of dissimilar
size (³
4 mm difference in diameter) had reduction compared to 19 of 26 (73%) with
vesicles of similar size (Ginther. 1989). As a result of
work studying reduction of unilateral versus bilateral twin pregnancies
in mares from days 17 to 40 Ginther
proposed a deprivation hypothesis. The deprivation hypothesis proposed that the
nutrient intake from the larger vesicle (pre foetal detection) prevented
adequate nutrition of the smaller vesicle. Later the position of the embryo
proper and it’s emerging allantoic sac seemed to determine whether a given
conceptus survived or underwent late reduction. The embryo proper, the
vasculised wall of the yolk sac adjacent to the embryo proper and the emerging
allantoic sac were exposed to the endometrium (uterine lumen) in the surviving
vesicles. In the vesicles that underwent reduction, much of the corresponding
area of the vesicle wall was covered by the wall of the adjacent survivor. Thus
embryo reduction occurs when a major portion of the three walled area of the
yolk sac or the vasculised wall of the yolk sac or allantoic sac is in
apposition with the wall of the adjacent vesicle rather than with the
endometrium and the vesicle is thus deprived of adequate embryonal-maternal
exchange and therefore regresses. In
summary, dissimilarity in diameter increased the likelihood of unilateral
fixation, increased the incidence of reduction for unilateral fixed vesicles,
hastened the day of occurrence of reduction and shortened the interval from
initiation to completion of reduction. The incidence of reduction for
bilaterally fixed embryos was negligible and approximates that of standard early
embryonic death in this period. Of the 85% of reductions by day 40 in cases of
unilateral fixed twin pregnancies, 59% of reductions had occurred between day 17
and 20, 27% between day 21 and day 30 and 14% between days 31 to 38. The
majority of early reductions occurred spontaneously (<20 days, or by day 20)
as compared to reductions after day 20 that were proceeded by a gradual decrease
in size of the eliminated vesicle. In addition when twins were dissimilar in
diameter (4 mm or more) they were more likely to undergo reduction by day 20
(Ginther. 1989). Other studies have demonstrated similar results. Examination of
69 sets of twins revealed, the greater the disparity in size, the greater the
chance of unilateral fixation (Ginther. 1987). Differences of greater than 3 mm
were associated with 83% unilateral fixation compared to 56% for less than 3 mm
(Ginther. 1984). The hypothesis of an early embryonic reduction mechanism for
elimination of an excess embryo(s) in mares was not new and had been suggested
as early as 1982. However, ultrasonography was necessary to adequately document
the occurrence and nature of the reduction (Ginther, Douglas, & Woods.
1982). Postfixation Day 40 onwards Although
many studies report the visible signs of later
twin pregnancies ie abortion, still-birth or production of live foals,
apparently only one study has documented (using ultrasonography) the outcome of
twin pregnancy after day 40 (Ginther & Griffin. 1994). Ginther and From
the above discussion it would appear that non intervention is only acceptable
when twins are diagnosed as a unilateral occurrence between days 17 and day 40
and depends on factors such as the value of the foal, the potential for
rebreeding and the ability of the veterinarian to manually intervene.
Intervention in twin pregnancies is strongly recommended in all other
circumstances (see below). Outcome of twin pregnancies 2) Intervention Prefixation Days 11-16 The
first technique for manual crush of the
conceptus during the mobility phase utilised manual reduction with good results
(Ginther. 1983) and was a variation from previously reported techniques for twin
pregnancies (Pascoe. 1979; Roberts. 1982). The technique involved gentle
manipulation of the embryonic vesicle to the tip of one uterine horn and manual
rupture. When applied to single pregnancies it resulted in pseudopregnacy and
when applied to twin pregnancies it resulted in a single pregnancy in 7 of 8
attempts (Ginther. 1983). Later (Pascoe, Pascoe, Hughes, Stabenfeldt, &
Kindahl. 1987; Pascoe, Pascoe, Hughes, Stabenfeldt, & Kindahl. 1987)
utilising the same techniques, mares were treated with single or multiple
progesterone administration, an anti-prostaglandin (flunixin meglumine) plus
progesterone or given no treatment prior to manual embryonic rupture in the
mobility phase. Results were 10/10 (100%) mares maintaining pregnancy in the
control group (no treatment, just manual rupture) and 37/40 (92.5%) for treated
mares. The amount of PGF2a
released was directly correlated with the pressure required to cause embryonic
rupture. Flunixin meglumine inhibited PGF2a
release after embryonic rupture. Treatment with progesterone plus flunixin
meglumine or progesterone singly or multiply was not better than no treatment at
all (Pascoe, Pascoe, Hughes, Stabenfeldt, & Kindahl. 1987; Pascoe, Pascoe,
Hughes, Stabenfeldt, & Kindahl. 1987), although it was subsequently shown
that the progesterone chosen (hydroxyprogesterone caproate) had no ability to
maintain pregnancy in ovariectomised mares and did not bind to progesterone
receptors in the horse (McKinnon, Figueroa, Nobelius, Hyland, & Vasey.
1993). Another report (Bowman. 1986) demonstrated that 60 of 66 mares (90.9%)
maintained a single vesicle after manual reduction was attempted prior to
fixation. Five of the six mares in which the procedure was not successful
subsequently conceived. Since 1984 we have used a modification (McKinnon, Voss,
Squires, & Carnevale. 1993) of the technique described originally (Ginther.
1983). With this technique the ultrasound probe is used to manipulate the
foetuses while keeping one or both foetuses in view during the manipulation and
more importantly the crushing or rupture of the vesicle. Utilising this
technique it is possible to more accurately and quickly separate vesicles. It
was original proposed (Ginther. 1983) that when vesicles were in apposition
mares be re-examined approximately one hour later. By utilising the probe to
manipulate vesicles, separation is achieved (pre-fixation) very quickly in most
instances. Commonly the smaller foetus is destroyed despite the lack of evidence
to support pre-fixation reduction. On occasion it is necessary to revisit the
mare 24-48 hr after the original evaluation if the smaller of the two vesicles
is less than 1 cm in diameter as sometimes these can be more difficult to
destroy. At the GVEH records were available for 522 Thoroughbred pregnancies
from the last breeding season. Eighty six (16.5%) had twins diagnosed
pre-fixation. After twin reduction mares are not routinely examined until the
next scheduled examination ie. 21-25 days post ovulation (detection of the
foetus). When mares were re-examined after pre fixation embryonic reduction all
were found to still be pregnant. The number of mares that originally had twins
loosing the remaining pregnancy prior to 45 days was 1/86 (1.2%), which was less
but not significantly different from 22/436
(5.0%) the normal singleton
population on these farms. It is our contention that the procedure has developed
to the stage that it is always expected a single pregnancy will exist after pre fixation
embryo reduction is attempted. Unless a mistake occurs and the other vesicle is
ruptured at the time of initial manipulation, we feel that any failure to
survive the procedure is more likely a result of uterine inflammatory changes
and infection rather than a result of the procedure. We believe that this is the
most reliable technique available but feel it is important to highlight the
experience of the personnel involved. From discussions with farm managers and
other veterinarians it is clear that only veterinarians involved with
sophisticated reproductive management such as the routine use of ultrasonography
can expect to achieve these types of results. Our strong recommendation to
veterinarians and clients is that all mares are examined within 14-16 days of
breeding. Expected time to ovulation after breeding will depend on ovulation
induction agents such as hCG or GnRH. Factors that may modify this decision are
breed, mare value, ability of the stud master or owner to facilitate examination
of the mare and on occasion education of the owner. Post fixation intervention Days 17-20 In
all cases of bilaterally fixed twins one is destroyed immediately, however the
mare has an extremely efficient biological embryo reduction mechanism that
operates when twins are in apposition (unilaterally fixed) (Ginther. 1984;
Ginther, Douglas, & Woods. 1982; Ginther. 1989). In one study the incidence
of embryo reduction after unilateral fixation was 14/19 (73.7%) which was
significantly greater than 0/9 (P<0.01) for bilateral fixation. In addition
asynchronous ovulation resulted in 90% (9/10) embryo reduction after unilateral
fixation. From the 14 mares that had embryo reduction, 10 (71.4%) had early
embryonic reduction (17-20 days) (Ginther. 1989). Other studies confirmed these
results. The incidence of reduction was 41/48 (85%) following unilateral
fixation (Ginther. 1989). Reduction occurred in 100% of 22 mares with
asynchronous ovulation (vesicles size greater than 4 mm in diameter) and 19/26
(73%) of mares with vesicle size (0-3 mm difference). Of all the reductions
occurring, 59% of the reductions occurred between 17 and day 20, 27% between day
21 and day 30 and 14% between day 31 to day 38. In the early reductions the
vesicles just simply disappeared. Reductions that occurred after day 20 were
preceded by a gradual decrease in size with the vesicle that was lost. As the
number of days after day 17 increase the frequency of reduction decreased and
the time required for completion of reduction increased. Because the rate of
embryo reduction between day 17 and
20 is so high for unilateral fixation, equine practitioners frequently elect to
leave these developing pregnancies and determine their outcome later. Our
philosophies are that if the two vesicles have coalesced into one larger vesicle
with an ultrasonographically visible line in division they are left totally
alone, however if the vesicles have still retained a spherical orientation or a
spherical shape, then they can be separated gently with the probe and are
crushed either in situ or after being manipulated apart. Due to the nature of
our practice, few mares present with this configuration in the thoroughbred
population (2-3 per year), however, it is not uncommon in the Standardbred
population wherein economics dictate that pregnancy diagnosis is often delayed
past the time the mobility phase has ended. Results from our
practice with twins in this configuration are reduced compared to
prefixation intervention procedures (8/12-75 %). Others have reported good
results post fixation. One group reported success in 49/50 cases post fixation
(Pascoe, Pascoe, Hughes, Stabenfeldt, & Kindahl. 1987). The work of Bowman
(1986) (Bowman. 1986) more closely parallels our experiences. With bilateral
embryo fixation and intervention, he reported almost no losses with 40/44 mares
from day 16 to day 30 (90.9%) having a single pregnancy detected on day 45. With
unilateral fixation the results were days 16 to 17 (16/18 - 89%) days 18 to 19
(23/24 - 95.8%) days 20 to 21 (8/13 - 61.5%) days 22 to 24 (4/9 - 47.4%) days 25
to 30 (1/4 - 25%). Because of the high incidence of
embryo reduction with unilateral fixation and the low incidence with
bilateral fixation, we have clear recommendations with twins in the days 17 to
20 period. Those that have rounded (figure ¥
shaped) twins, still retaining their vesicle turgidity, that can be separated,
are crushed either in situ or after being manipulated apart. In all cases where
the vesicles have apparently coalesced into a larger vesicle with an
ultrasonographically single line dividing the two we leave them alone, more
particularly so, if there is any unevenness in vesicle size. In all cases of
bilaterally fixed twins one is always destroyed immediately. Day 21 to 30 All
cases of bilaterally fixed twins of this age group are manipulated and one
destroyed immediately. In most cases
we do not attempt to manually destroy one vesicle with unilaterally fixed twins
of this age group until after day 30 and before day 35. At this age it is too
easy to rupture both vesicles and the maximum success we believe we can expect
is 50% (see previous section) which is less than or similar to the mares own
biological reduction mechanism. Day 30 to 35 During
the period prior to the formation of endometrial cups, gentle pressure may be
placed on one vesicle. We do not attempt total ablation at this time as
resulting fluid sometimes surrounds the other foetus and effectively separates
placental (chorionic girdle / trophoblast cells) attachments to the uterus. In
these cases (total rupture of the vesicle), death of the remaining vesicle is
very common. Between day 30-35 we attempt to pinch one vesicle and create a
“snow flake” effect which is the shedding of cells from the membranes.
Demonstration of this effect almost always results in gradual loss of the
effected conceptus Day 36 to 60 From
day 36 onwards it is a reasonable assumption that endometrial cup formation and
subsequent eCG secretion will prevent many mares from returning to heat after
early embryonic death. Abortion after 35 days is commonly associated with
difficulties recycling the mare (Baucus, Squires, Morris, & McKinnon. 1987;
Squires & Bosu. 1993). In one study (Penzhorn, Bertschinger, & Coubrough.
1986) when mares were aborted either between day 26 and 31 or between day 30 and
50, 8/11 became pregnant versus 2/7, respectively. This is similar to the work
of (Pascoe. 1983) who concluded that the administration of a prostaglandin
analogue < 35 days of gestation
was outstandingly successful as a method of treatment for twin pregnancy. Manual
intervention at this time in our experience is approximately 50% successful.
Success improves with use of more subtle pressure and damage to the
chorioallantoic membrane rather than complete rupture in one attempt.
Demonstration of the ‘snowflake
effect’ without vesicle rupture consistently results in a gradual (48 hr)
stress of the foetus and ultimate loss of heartbeat for the conceptus. These
pregnancies have the foetal fluids that become progressively more hyperechoic
and reduce in size without interfering with the survival of the other foetus. It
is important with these foetuses to always attempt to damage the same one.
Multiple attempts, ie everyday or every other day for 5 to 10 sessions maybe
necessary to elicit the correct response, however, quite frequently we are
unable to create sufficient damage for foetal destruction. In these cases rather
than creating major trauma (rupture of the vesicle) an alternative approach is
sought after day 60. A variety of methods have been used to treat twins at this
stage. Manual crushing was originally reported (Roberts. 1982) and results
suggested that earlier crushing was better and if possible crushing should occur
prior to day 31 because after day 35 sometimes manual rupture was not possible.
The author quoted the following results between day 35 and 45; 60%
resorption of both 20% single foaling 20% survival of both . One author
(Pascoe. 1983) concluded recycling with prostaglandin prior to day 35 was an
outstandingly successful method of treatment. This same author demonstrated that
needle puncture of one twin combined with nonsteroidal antinflammatory treatment
(meclofenmic acid) resulted in no foals born. A more elaborate and invasive
approach, such as intra-foetal injection with saline via a laparotomy has been
reported (Hyland, Maclean, Robertson-Smith, Jeffcott, & Stewart. 1985) or
removal of one foetus via a video endoscope (Allen & Bracher. 1992)
(abandoned as being non practical). An interesting report was the surgical
technique for removal of one conceptus from mares with twin concepti more than
35 days of gestational age (Stover & Pascoe. 1987; Pascoe & Stover.
1989). Eight mares had bicornuate pregnancies and 7 mares had unicornuate twin
concepti. Five of six surviving mares with bicornuate twin concepti, delivered a
single viable foal and none of the 7 mares originally with unicornuate twin
concepti, produced a foal. The poor survival rate of unicornuate twin concepti
was attributed to disruption of the remaining chorioallantois during surgery.
13/14 mares are reported to have been successfully rebred. Transvaginal
ultrasound guided foetal puncture for destruction of one of a set of
twin pregnancies has been reported (Bracher, Parlevliet, Pieterse, et al.
1993). Foetal fluids from one foetus were aspirated while observing the
relationships of the needle foetus yoke sac and/or allantochorion between days
20 and 45. Three of four bicornuate between pregnancies resulted in
a single pregnancy 10 days or greater
after interference (similar to our ability to manually destroy one
conceptus in this configuration ).
Three of nine (33%) still had a viable single pregnancy after 10 days when twins
were fixed together ( between day 20 and 45). These results were disappointing,
however they maybe improved with experience and/or antibiotic therapy at the
time of intervention. Ultrasound guided fluid withdrawal between day 50 and 65
was studied in single pregnancies (Squires, Tarr, Shideler, & Cook. 1994)
however, the study did not involve any twins. Our experiences with transvaginal
ultrasound guided foetal reduction are small (N=5) however, between 45 and 60
days the foetus within the vesicle was difficult to position. We only have
attempted to directly puncture the foetus, not aspirate fluid and are unlikely
to persevere with this technique ( foetal puncture at this age ) due to
difficulties involved. All cases ended in loss of both foetuses, usually within
three days of interference. Day 60 to 100 Between
day 60 and 100 it becomes more difficult to damage the chorioallantois. In these
cases we identify the most
conveniently located (always the smallest) of the twins and repeatedly
traumatise it by oscillation or attempt to damage the cranium with multiple
attempts of single digit percussion. Similarly to the previous scenario
approximately 50% succumb to this procedure, however it is tedious and time
consuming. Days 100 onwards Probably
the most common reason for being presented mares at this late stage of gestation
with twins is failure of the aforementioned techniques. Less frequently twins
have been missed in earlier diagnostic attempts. Frequently mares have been
identified with twins late in the breeding season and the owner has adopted a
non-intervention approach. Because
the possibility of foetal reduction after 100 days is very low and the
probability of abortion or stillbirth is extremely high, an approach was
developed to eliminate one pregnancy at a later stage of gestation (Rantanen
& Kincaid. 1988). The technique involved transabdominal ultrasonographic
identification of the twins and intracardiac injection of a lethal substance.
The smaller twin was always identified. Initial results with saline and air were
unsuccessful but when the solution was replaced with potassium chloride, 7/18
mares (39.9%) had single live foals this rate has subsequently improved. We have
been utilising this technique since 1988 and can report similar experiences.
Initial success was not very promising (2/10 live foals) until the potassium
chloride solution was replaced with 10ml-20ml of
procaine penicillin which has resulted in 5 live foals from the last 9
mares attempted (with the opportunity to foal). The current procedure at the
GVEH is to tranquillise the mare
with Detomidine (McKinnon, Carnevale, Squires, & Jochle. 1988) and to
identify the smaller foetus or in the case of evenly sized foeti the one most
accessible. A 6 inch, 16 or 18 gauge needle with a tip designed for
ultrasonographic enhancement ( From
all of the preceding discussion it
should be obvious to readers that in our opinion the best method of handling
twins is early identification and destruction of one (day 11 to 16). Early
Embryonic Death Early
embryonic death results in low reproductive performance of mares. Notifying the
client that a valuable mare has undergone embryonic loss is an annoying
experience for a breeding manager or veterinarian. Clients enthusiastically
support use of ultrasonography for early pregnancy detection, however, not all
pregnancies continue to survive, even in normal mares. Improvement in
ultrasonographic equipment has permitted investigation of early embryonic losses
between days 10 and 20 of gestation (Villahoz, Squires, Voss, & Shideler.
1985). This technique, combined with embryo recovery, permits investigation of
embryo losses between day 6, which is the first time an embryo can be routinely
recovered from the uterus, and day 11, which is the first time the vesicle can
be consistently detected by ultrasonography. The real incidence of EED prior to
day 6 is unknown. However, it was suggested that a major proportion of EED in
infertile mares occurred in the oviduct (Ball, Little, Hillman, & Woods.
1986). More recently workers in Early
embryonic death is diagnosed when an embryonic vesicle seen previously is not
observed on two consecutive ultrasonographic scans and/(or) when only remnants
of a vesicle are observed. Ultrasonographic criteria for impending EED are an
irregular and indented vesicle, fluid in the uterine lumen and vesicular fluid
that contains echogenic spots. Early embryonic death is suspected, particularly
after day 30, when no foetal heartbeat is observed, there is poor definition of
foetal structure, foetal fluids are very echogenic, or the largest diameter of
the foetal vesicle is two standard deviations smaller than the mean established
for that specific day of age. Vesicles increasing in size more slowly than
normal may also be characteristic of early embryonic death (Ginther, Bergfelt,
Leith, & Scraba. 1985). Indications obtained by ultrasonographic scanning of
impending loss at later stages include: failure of fixation, an echogenic ring
within the vesicle, a mass floating in a collection of fluid and a gradual
decrease in volume of placental fluid with disorganisation of placental
membranes. Ultrasonographic scanning, during early pregnancy, is an extremely
useful management tool for pregnancy detection and determination of early
embryonic death. However, if pregnancy rates are not reported until day 50, the
discrepancy between pregnancy and foaling rates decreases. There are few, if
any, treatments to consistently decrease incidence of EED, but artificial
insemination can limit bacterial challenge to a mare's uterus, thus reducing
potential losses from endometritis. In addition, any new information on causes
and treatment of endometritis should result in increased breeding efficiency.
The transfer of embryos from mares with poor uterine-biopsy grades into normal
recipient mares is recommended to provide an environment more conducive to
pregnancy maintenance. Unfortunately, recovery of embryos from infertile mares
is low. Supplementation with progesterone to habitually aborting mares or mares
with primary luteal inadequacy has been advocated. However, there is little
experimental evidence on the efficacy of this procedure (Allen. 1984) and
another suggestion some of the progestagens available do not even work
(McKinnon, Tarrida del Marmol Figueroa, Nobelius, Hyland, & Vasey. 1993). A
recent report suggests genetic abnormalities would not appear to be a major
cause of EED in mares (Romagnano, Richer, King, & Betteridge. 1987). Perhaps
the changes most likely to result in a decrease in incidence of EED is improving
management factors related to nutrition, environmental temperature, infectious
diseases and other stresses. Foetal
Monitoring A
disproportionate interest has been directed towards conception, early pregnancy
diagnosis and the foaling process. Most veterinarians are ignorant of the
development of the foeto-placental unit. Prenatal death although a major source
of reproductive wastage is often put in the “too
hard basket” or is left as unclassified abortion. Initial treatment of the
sick neonate is often delayed because delivery was unattended, neonatal
compromise was not recognised, or critical care was unavailable or not
economically feasible (Vaala & Sertich. 1994). Foals surviving severe
peripartum illness often experience increased morbidity associated with chronic
infection, developmental musculoskeletal disease and suboptimal growth. Focus
has shifted from a strictly therapeutic approach to prevention (Vaala &
Sertich. 1994). At the GVEH we are beginning to explore ways to assess foeto-placental
well-being during the latter stages of pregnancy and are aiming to identify
mares at risk for an abnormal pregnancy and/or delivery. Close supervision of
such cases during the periparturient period would allow earlier detection,
treatment and possible prevention of conditions affecting the mare or the foal.
Many techniques to monitor the equine foetus are available such as foetal
electrocardiography, biochemical indices of function such as measurement of
oestrogens, progestagens, equine foetal protein and placental fluid analysis and
non-invasive analysis of the pregnancy such as ultrasonography. The
equine foetus can be imaged with both transrectal and transabdominal
ultrasonography. The latter approach is most commonly employed in late
gestation, because of the close proximity of the foetus to the ventral abdominal
wall. In addition, because a complete examination requires 15 to 30 minutes, the
transabdominal approach is safer (for the
mare !). The mare is restrained in stocks and the ventral abdomen is clipped
or shaved, coupling gel is applied to the abdomen and the transducer. Sedation
is avoided owing to possible suppression of foetal heart rate (FHR) and foetal
activity (McGladdery & Rossdale. 1991). Transabdominal scanning can be
performed with the same equipment used for routine rectal reproductive
ultrasound examination, but preferably using lower frequency transducers (2.5 to
5 MHz), allowing deeper penetration and more complete examination. Both linear
array and sector scanners may be used; the primary advantage of the latter are
ease of directing the beam and ability to image deeper structures through small
acoustic windows. In the pregnant mare, transabdominal ultrasonography has been
used to detect twins, document foetal position, estimate foetal size, evaluate
placental integrity and foetal fluid clarity and gain a general appreciation of
foetal well-being. Recent studies have focused on the development of a modified
equine biophysical profile (BPP), using FHR reactivity, foetal activity, foetal
breathing movements, qualitative and quantitative assessment of foetal fluids,
evaluation of placental integrity and measurement of foetal size (Vaala &
Sertich. 1994). The foetal fluids are recycled several times a day via placental
and transcutaneous exchange, foetal swallowing and foetal urine production.
Oligohydramnios is an important sign of foetal asphyxia. Decreases in amniotic
fluid have been associated with dysmaturity, chronic intrauterine stress and
hypoxia, and placental insufficiency. During asphyxia, the most complex foetal
activity, foetal heart reactivity, disappears first, followed sequentially be
foetal breathing, foetal movements and, finally, foetal tone. During chronic
hypoxia, there is a reflex redistribution of foetal cardiac output in an attempt
to preserve blood flow to the foetal brain, heart and adrenal glands at the
expense of perfusing other organs, including the kidneys. As flow to the kidneys
diminishes, renal function decreases and production of foetal urine declines.
Decreased amniotic fluid volume may predispose to acute hypoxic episodes as a
result of abnormal umbilical cord compression during foetal movement and uterine
contraction. The maximum vertical fluid pocket depth of amniotic and allantoic
fluids range from zero around the caudal aspects of the foetus, to an average of
8 cm for amniotic fluid and 13 cm for allantoic fluid (Vaala & Sertich.
1994). Depth and location of fluid pockets change in response to foetal
movement. Excessive foetal fluid accumulation is observed in cases of hydrops
allantois or amnii. Increase in echogenic particles are observed in most foetal
fluids during advancing gestation and are associated with the presence of normal
vernix and urinary salts. Particle visibility increases after vigorous foetal
movement, which induces swirling of the fluids. Sudden increases in turbidity
may be associated with the passage of meconium in utero, haemorrhage or
inflammatory debris and may reflect foetal hypoxia, placental detachment or
placental infection (Vaala & Sertich. 1994). Detailed
sonographic studies during late gestation reveal most foetuses in an anterior
presentation, lying in an oblique ventrodorsal position (slightly tilted on its
back). In early gestation (60-120 days), the transducer may need to be placed in
the inguinal areas to image the foetus, while in late gestation, the foetus
may extend as far cranially as the xiphoid. Ultrasonography in late
gestation can provide assurance that the foal is in proper presentation for
delivery. Inconsistencies
in ages determined from history and the sonagram, may indicate intrauterine
growth retardation, caused by developmental anomalies or placental
insufficiency. Chronic placental insufficiency frequently manifests itself as in
utero foetal growth retardation, which in turn increases foetal risk of
perinatal morbidity and mortality. In utero measurements of various foetal body
parts, have resulted in elaborate in utero growth charts for the human foetus,
at various stages of gestation. The large size of the equine foetus in late
gestation, precludes using large body part measurements to establish an accurate
in utero growth chart. Structures which have been measured sequentially and for
which a correlation with neonatal weight has been established, include foetal
orbit size (Kahn & Leidl. 1987; McKinnon, Voss, Squires, & Carnevale.
1993) and foetal aortic diameter (Adams-Brendemuehl & Pipers. 1987). Foetal
orbit is often difficult to visualise, especially in late term mares, using a
transabdominal approach. Foetal aortic diameters, measured in systole in
thoroughbred mares in the week before parturition, correlated well with weight,
circumference at the girth an hip height of newborn foals. Aortic diameter
increased from 2.1 cm at 300 days gestation, to 2.7 cm at term in foetuses
examined serially; consequently, aortic diameter increases at about 1 mm per 5
days in late gestation (Adams-Brendemuehl & Pipers. 1987). Similar measures
of systolic aortic diameter performed in Arabian and draft breed foetuses,
revealed breed variation. Unfortunately, aortic diameter measured during foetal
ultrasonography of high risk pregnancies, correlated poorly with birth weight.
In addition, in many pregnancies which resulted in the delivery of emaciated or
dysmature foals, transabdominal ultrasonographic measurement of aortic diameter
was normal. Consequently, before transabdominal ultrasonography becomes useful
for the determination of foetal age or size in horses, further foetal
morphometric studies should be
carried out, in conjunction with ultrasonographic measurements to identify
appropriate anatomical structures to measure. Foetal
breathing is characterised by excursions of the diaphragm between the thorax and
the abdomen, accompanying rib cage expansion, Foetal breathing movements are
observed in most late term equine foetuses, when the foetal diaphragm can be
visualised. Care must be taken not to mistake movement associated with
maternal breathing with foetal respiration. During
late gestation, the equine foetus should demonstrate good tone and moderate
activity with only short periods of inactivity (<10 minutes). In general,
equine foetal activity increases with advancing gestational age. In contrast,
foetal heart rate decreases with approaching parturition. During the last month
of gestation, FHR averages between 70-90 beats/min. (bpm) (Pipers & Adams-Brendemuehl.
1984; Vaala & Sertich. 1994); transient bouts of tachycardia are observed
during or immediately after foetal activity, with an expected increase above
resting baseline of 25-40 bpm. Foetal cardiac rhythm should be regular.
Persistent bradycardia is associated with varying degrees of foetal distress and
is mediated by a vagal response to hypoxemia. Severe foetal tachycardia and
arrhythmia’s have been associated with impending foetal demise (Vaala &
Sertich. 1994). The responsiveness of heart rate during movement is a more
sensitive indicator of foetal distress than single heart rate recordings
obtained with the foetus at rest or following activity. Normal equine foetal
behaviour states during late gestation have not been characterised and would
require continuous 24-hour rate and foetal movement monitoring. In general, the
equine foetus appears to spend less time asleep than the human foetus and
periods of inactivity exceeding 15-20 minutes, should be investigated. The
average uteroplacental thickness, ranges between 7 and 13 mm (Vaala &
Sertich. 1994). A thicker
uteroplacental unit may be associated with placenta oedema, impending premature
placental separation, or placentitis. Placental oedema has also been seen with
fescue toxicity (Green, Loch, & Messer. 1991). Areas of separation between
chorion and uterus appear as anechoic spaces. Small areas of separation normally
appear at the site of umbilical vessel attachment. Large or progressively
enlarging areas of placental detachment are abnormal and contribute to foetal
compromise and death (Cottrill, Jeffers-Lo, Ousey, et al. 1991). When such
changes are observed, induction of parturition should be considered to reduce
the risk of foetal hypoxia or death. Doppler
velocimetry. Doppler ultrasonography is the newest diagnostic tool being used in
humans to examine foeto-placental circulation in an attempt to identify
pregnancies at risk for foetal intrauterine growth retardation. Although some
preliminary work has been conducted in pregnant pony mares, more baseline data
need to be collected on normal equine gestations, before the technique can be
effectively used in the evaluation of high risk pregnancies (Vaala & Sertich.
1994). UTERINE
PATHOLOGY With
ultrasonography the uterus can be examined non-invasively for pathologic changes
and to monitor therapeutic regimen(s). The three most common forms of uterine
pathology detected by ultrasonography are accumulations of intrauterine fluid,
air and cysts. Less commonly, foetal remnants, debris, abscessation and
neoplastic conditions are observed. Intrauterine
Fluid Ultrasonography
is extremely valuable for estimating quantity and quality of fluid in the
uterine lumen. Rectal palpation is only accurate when quality of intrauterine
fluid is large (> 100 ml) and(or) when uterine tonicity changes. Confirmation
of intrauterine fluid, without invasive techniques such as lavage and
cytological analysis, was difficult until direct, non-invasive visualisation was
made possible with ultrasonography. Volumes of fluid within the uterine lumen
are estimated with ultrasonography and quality is graded from I to IV according
to degree of echogenicity (McKinnon, Squires, Harrison, Blach, & Shideler.
1988). Degree of echogenicity is related to amount of debris or white blood cell
infiltration into the fluid. Grade I fluid has large numbers of neutrophils and
grade IV has very few neutrophils. Observations on quality and quantity of
uterine fluid have been used to assess efficacy of various therapeutic
procedures on individual animals treated for naturally occurring endometritis.
Experiments (McKinnon, Squires, Harrison, Blach, & Shideler. 1988; McKinnon,
Squires, Carnevale, et al. 1987; Adams, Kastelic, Bergfelt, & Ginther. 1987)
have been conducted to determine the relationship of intrauterine fluid to
fertility. Ultrasonographic
Studies of the Uterus After Parturition In
the equine industry, economic incentives influence breeders to attempt a foaling
interval of 12 Mo or less. This
commonly necessitates breeding of mares during the first post-partum ovulation.
However, fertility has been reported lower in mares bred during the first
post-partum ovulatory period compared with mares bred during subsequent cycles (Merkt
& Gunzel. 1979), and early embryonic death has been reported higher for
mares bred at this time (Lieux. 1980; Merkt & Gunzel. 1979; Platt. 1973).
This decreased fertility may be due to failure of elimination of microbes during
uterine involution (Merkt & Gunzel. 1979; Platt. 1973) or their introduction
at breeding. In addition, presence of uterine fluid during oestrus (McKinnon,
Squires, Harrison, Blach, & Shideler. 1988) and dioestrus (McKinnon,
Squires, Carnevale, et al. 1987; Adams, Kastelic, Bergfelt, & Ginther. 1987)
has been shown to reduce fertility of mares. A study (McKinnon, Squires,
Harrison, Blach, & Shideler. 1988) was conducted to evaluate two hypotheses:
1) uterine involution and fluid accumulation could be effectively monitored with
ultrasonography and used to predict fertility of mares bred during the first
post-partum ovulatory cycle, and 2) delaying ovulation with a progestin would
result in improved pregnancy rates in mares bred during the first post-partum
ovulatory period. The previously gravid horn was larger than the non-gravid horn
for a mean of 21 days (range 15 to 25) after parturition. Uterine involution was
most obvious at the corpus cornual junction. When the results of three
ultrasonographic scans were similar, over a 5-day period, the uterus was
considered to be involuted. On the
average, uterine involution was completed by day 23 (range 13 to 29). Quantity
and quality of uterine fluid were not affected by progestin treatment. Number of
mares with detectable uterine fluid decreased after day 5 post-partum. Uterine
fluid generally decreased in quantity and improved in quality between days 3 and
day 15. Fewer (P < 0.005) mares became pregnant when uterine fluid was
present during the first post-partum ovulatory period (3 of 9, 33%), compared to
when no fluid was detected (26 of 31, 84%). Mares with uterine fluid during
breeding did not have appreciably larger uterine dimensions, compared with those
mares not having fluid. There was no relationship between uterine size on day of
ovulation and pregnancy rate. Ovulations were delayed, and pregnancy rates
improved in progestin-treated mares. More (P < 0.05) mares became pregnant
(23/28, 82%) when they ovulated after day 15, in the first post-partum ovulatory
period, than mares that ovulated before day 15 (6/12, 50%). Ultrasonography has
been proven useful in detecting mares with post-partum uterine fluid. Further,
it could be used to aid in determining whether a mare should be bred, treated,
or not bred during the first post-partum ovulatory period. During oestrus,
uterine fluid may be spermicidal and/(or) an excellent medium to support
bacterial proliferation. When fluid is present during dioestrus, it may cause
premature luteolysis or early embryonic death (Adams, Kastelic, Bergfelt, &
Ginther. 1987). Quantity of uterine fluid during the first post-partum ovulatory
period appeared to be related to stage of uterine involution, and was reduced or
eliminated by delaying the ovulatory period with progestins. Progestin treatment
not only allowed time for elimination of uterine fluid before the first
post-partum ovulation, but it also significantly delayed the first post-partum
ovulation. Results of this study concurred with those of others in which it was
concluded that progestin treatment delayed onset of the first post-partum
ovulatory period, but did not affect rate of uterine involution (Loy, Evans,
Pemstein, & Taylor. 1982; Pope, Campbell, & Davidson. 1979; Sexton &
Bristol. 1985). Long-term progestin administration to normal, cycling mares has
not been shown to adversely affect fertility (Squires, Heesemann, Webel,
Shideler, & Voss. 1983). However, treatment with progestins will affect
uterine defence mechanisms (Evans, Hamer, Gason, Graham, Asbury, & Irvine.
1986; Winter. 1982) and thus care is recommended before prolonged progestin
treatment is administered to post-partum mares or mares susceptible to
infection. Since there were decreased pregnancy rates associated with uterine
fluid, and increased pregnancy rates as ovulation was delayed, it was suggested
both techniques could be used to manipulate breeding strategies and improve
pregnancy rates from normal mares bred during the first post-partum ovulatory
period. Effect
of Intrauterine Fluid on Pregnancy Rate and Early Embryonic Death. A
study was designed to determine the influence of intrauterine fluid on pregnancy
rate and early embryonic death (McKinnon, Squires, Carnevale, et al. 1987). It
was concluded from this study that: 1) presence of intrauterine fluid during
oestrus in cycling mares did not affect pregnancy rates at either day 11 or 50;
2) intrauterine fluid, detected 1 or 2 days after ovulation, did not affect
day-11 pregnancy rates, but was associated with a significant increase in EED
and reduced day-50 pregnancy rates; and 3) presence of intrauterine fluid during
dioestrus (days 1 to 20 post-ovulation) was associated with a significant
decrease in day-50 pregnancy rates. At the GVEH the amount and quality of fluid
detected is the determinant of how to treat the mare. Large volumes of poor
quality (grade 1 or 2) fluid are treated with voluminous saline lavage, whilst
small volumes of grade 4 fluid quite often are treated with local antibiotics
and the mare bred the next day.
Further information may be obtained in the chapter on breeding the problem mare. Diagnosis
of Endometritis. There
are numerous techniques to diagnose endometritis.
However, no technique is completely reliable. The common, currently
accepted techniques are: 1) rectal palpation, 2) vaginal-speculum examination,
3) bacterial culture of uterine contents, 4) cytological examination of uterine
contents, 5) endometrial biopsy and 6) ultrasonography. A study was conducted
(McKinnon, Squires, Carnevale, et al. 1987) to examine the efficacy of
individual diagnostic techniques to predict endometritis.
This study demonstrated that ultrasonography was as accurate as all the
other diagnostic tests of endometritis. In addition, it was determined that in
progesterone dominated mares, multiple invasive procedures (ie. culture, biopsy,
vaginal-specular examination and cytologic specimen collection) resulted in
persistent endometritis, thus highlighting the usefulness in having a
non-invasive diagnostic test such as ultrasonography. Uterine
Cysts Prior
to ultrasonography, uterine cysts were most commonly diagnosed from post-mortem
examination and occasionally by rectal palpation (Kenney & Ganjam. 1975).
More recently they have been diagnosed by hysteroscopy and ultrasonography
(McKinnon, Squires, Carnevale, et al. 1987; McKinnon, Squires, & Voss.
1987). Cysts in the uterus are fluid-filled and apparently have two origins. The
histological structures of uterine cysts have been described. Endometrial cysts
arise from endometrial glands, and are usually < 10 mm in diameter. Their
incidence and significance is largely unknown. The second form of uterine cysts
are lymphatic in origin and generally are larger than endometrial cysts. They
are common in older mares (Adams, Kastelic, Bergfelt, & Ginther. 1987), and
have been associated with both normal and abnormal uterine biopsies (Kenney
& Ganjam. 1975). Size of uterine cysts may be indicative of origin. No data
has been reported on growth rate of uterine cysts. Despite the occasional large
cysts reported, it is unlikely that they grow at a similar rate as the early
embryonic vesicle (days 10 to 20). When visualised with ultrasonography, cysts
are commonly rounded, with irregular borders, and occasionally are multiple or
compartmentalised. Movement of the early equine conceptus (days 10 to 16),
presence of specular reflection, spherical appearance and growth rate of the
embryo should aid in its differentiation from uterine cysts. The relationship
between infertility and uterine cysts is axiomatic. Cysts may impede movement of
the early conceptus, restricting the reported ability of the vesicle to prevent
luteolysis after day 10 (McDowell, Sharp, Grubaugh, Thatcher, & Wilcox.
1988). Later in pregnancy, contact between the cyst wall and yolk sac or
allantois may prevent absorption of nutrients. This may be more important when
considering the recognition that large uterine cysts are more commonly located
at the junction of the uterine horn and body, which is the most common site of
vesicle fixation (Ginther. 1983). Finally, cysts are commonly indicative of
uterine disease. They may reflect senility or be associated with endometritis.
It has been reported (Adams, Kastelic, Bergfelt, & Ginther. 1987) that there
is an association between number of uterine cysts, age of mare, and endometrial
biopsy. The number of treatments proposed for uterine cysts probably reflects
inability of any individual treatment to consistently be useful. Rupture of the
fluid-filled structures has been attempted via uterine-biopsy forceps (Kenney
& Ganjam. 1975), surgery, fine needle aspiration and puncture via
hysteroscopy. Electro-coagulative removal of cysts has also
been described. Endometrial curettage and repeated lavage with warm
saline (40 to 450C) have also been advocated (Kenney & Ganjam.
1975). Although there are no reports on respective efficiency of these
treatments, endometrial curettage and saline lavage are frequently applied to
treat the primary problem, which would appear to be lymphatic blockage. It was
concluded from one study that: 1) uterine cysts, when detected by
ultrasonography, were lymphatic in origin; 2) uterine cysts did not change
rapidly in size or shape, although they were more difficult to detect during
oestrus; 3) treatment with infra-red radiation was not effective; 4) there was
no consistent location for uterine cysts; and 5) uterine cysts were commonly
associated with chronic, infiltrative, lymphocytic endometritis (McKinnon,
Squires, Carnevale, et al. 1987). Miscellaneous
Uterine Pathology Recently
we have identified other less commonly recognised forms of uterine pathology,
the most common of which was air in the uterus. Air is recognised as multiple,
hyper-echogenic reflections (occasionally a ventral reverberation artefact is
present) and it appears to be more prevalent slightly cranial to the cervix,
although it can be present in the cranial body or uterine horns. Air, when
present < 24 hr after artificial insemination, is considered normal. However,
it is not expected to be found in normal mares < 48 hr after breeding. The
observation of air in the uterus of mares that have not been bred recently is an
indication of pneumo-uterus and reflects failure of the competency of the
vaginal labia, vestibulo-vaginal sphincter and(or) cervix (McKinnon, Squires,
Carnevale, et al. 1987). On occasion, strongly echogenic areas in the uterine
lumen are observed with a concomitant echo shadow, such as is seen with dense
tissue like foetal bone. This might be expected after mummification. We have
also identified a similar ultrasonographic image that was confirmed subsequently
as the tip of a uterine culturette. Undoubtedly there are many other forms of
less commonly recognised uterine pathology such as uterine neoplasia, abscesses
and haematomas that will be recognised as ultrasonography of the uterus becomes
more routine. FOLLICULAR
DYNAMICS PRECEDING AND DURING OVULATION Ultrasonography
is useful for monitoring dynamic follicular and luteal changes in equine
ovaries, since it permits rapid, visual, non-invasive access to the reproductive
tract. A 5 MHz transducer has greater resolution and is more suitable for
evaluation of ovaries than a 3 or 3.5 MHz transducer. Follicles as small as 2 to
3 mm can be seen and the CL can usually be identified throughout its functional
life (Pierson & Ginther. 1985). Potential applications of ultrasonographic
examination of the ovaries include: 1)
estimating stage of the oestrous cycle, 2) assessing preovulatory follicles, 3)
determining ovulation, 4) examining the CL, and 5) diagnosing ovarian
abnormalities and pathology. Stage
of the Oestrous Cycle Follicles,
like other fluid-filled structures, are non-echogenic and appear as black,
roughly circumscribed ultrasonographic images. Compression by adjacent
follicles, luteal structures or ovarian stroma occasionally can result in
irregular-shaped follicles. The
apposed walls of adjacent follicles are often straight. Diameter can be
estimated by adjusting an irregular-shaped follicle to an approximately
equivalent circular form. Sequential monitoring of dynamic changes in a
follicular population during the oestrous cycle has been made possible by
ultrasonography. During anoestrus, inactive
ovaries are readily differentiated from functional ovaries with ultrasonography.
Occasional small follicles (2 to 5 mm) may be present but absence of an
ultrasonographically visible CL is characteristic of anoestrus. Multiple, large
follicles characteristic of transitional mares, prior to their first ovulation
of the year, are particularly frustrating to practitioners and researchers.
Generally, follicular atresia and subsequent growth occurs until one follicle
becomes dominant and ovulates. During transition, some ovulations are difficult
to detect by palpation and in these cases ultrasonographic observation of a CL
may confirm whether the mare has entered the ovulatory season. With the use of a
5 MHz transducer, the CL should be ultrasonographically visible for
approximately 14 days after ovulating (Pierson & Ginther. 1985). Examination
with ultrasonography has resulted in confirmation of the presence of many 5 to
10 mm follicles during early dioestrus, growth of large follicles at mid cycle,
observation of selective, accelerated growth of an ovulatory follicle beginning
6 days before ovulation, and regression of large non-ovulatory follicles a few
days before ovulation (Pierson & Ginther. 1985; Pierson & Ginther.
1985). Ultrasonographic examination of the ovaries should not replace sound
management techniques such as regular teasing, and rectal palpation to determine
stage of oestrous cycle, rather, it should be used as a powerful ancillary aid. Preovulatory
Follicles The
ability to accurately detect time of ovulation has significant practical
application. Selective growth of a single preovulatory follicle is initiated
about 6 days before ovulation (Pierson & Ginther. 1985). Various
characteristics can be used, within certain limitations, to predict time of
ovulation. Softening of the follicle commonly occurs within 24 hr of ovulation
in approximately 70% of mares. Ultrasonographically, this is frequently
associated with a change in follicular shape from spherical to pear or irregular
shapes (Pierson & Ginther. 1985), which may be due to disruption of ovarian
stroma as the follicle progresses toward the fossa in preparation for ovulation.
The mare's ovary is structurally inverted in comparison to most species with the
exception of the ovulation fossa, which is a 0.5 to 1 cm depression on the
lesser curvature. The tunica albuginea and mesovarium forms a thick serosal
coating covering the ovarian surface. Connective tissue tracts extend from the
ovulation fossa to the periphery, which forces the follicle to grow centrally
toward the fossa. These structural arrangements restrict ovulation to the
ovulation fossa. Cinematographic and histologic studies have been used to
determine the exact location of follicular rupture. However, time sequence and
follicular changes during ovulation are not well-characterised. Although
stallion semen has been reported to survive for up to 5 days or longer in the
mare's reproductive tract (Pickett, Squires, & McKinnon. 1987), it is more
commonly accepted that a lapse before ovulation of > 48 hr between breeding
will result in decreased numbers of viable spermatozoa and reduced fertility
(Pickett, Squires, & McKinnon. 1987). Use of frozen, cooled or poor quality
semen may markedly hinder life span of spermatozoa after insemination. Although
no critical studies have been performed, the mare's oocyte probably begins to
lose viability within 12 to 24 hr after ovulation (Pickett, Squires, &
McKinnon. 1987; Woods, Bergfelt, & Ginther. 1990). In addition, semen
deposited in the uterus after ovulation requires time to reach the oviduct (site
of fertilisation) and for capacitation. Breeding or insemination, particularly
with semen of reduced longevity, just prior to ovulation would maximise
pregnancy rates and prevent overuse of an individual stallion. Accurate
prediction of impending ovulation would allow for collection of mature equine
oocytes for in vitro fertilisation or for gamete transfer from infertile mares
(McKinnon, Wheeler, Carnevale, & Squires. 1986; McKinnon, Carnevale,
Squires, Voss, & Seidel, Jr. 1988). In addition, recently ovulated oocytes
or early cleavage embryos could be recovered from the oviduct at specific times
post-ovulation. In one study (Pierson & Ginther. 1985), various criteria
such as percentage change in shape, size of follicle, echogenicity of follicular
fluid and wall, and thickness of follicular wall were evaluated in their ability
to predict time of ovulation. Size of the preovulatory follicle was as accurate
as any method in determining ovulation time. Generally, double, preovulatory
follicles ovulated after attaining a smaller maximum diameter than single,
preovulatory follicles. Thickening of the follicle wall occurs in most
preovulatory follicles prior to ovulation. However, it generally occurs too
early to be an adjunct to predicting ovulation. Increased echogenicity of
follicular fluid is sometimes seen prior to ovulation, perhaps due to
degeneration and subsequent shedding of granulosa cells from the follicular
wall. This can be an indicator of impending ovulation, although it is neither
common or consistent enough to be particularly diagnostic. In general, the
combination of softening of a large follicle, particularly when associated with
pain as determined by rectal palpation, and a substantial change in shape of the
follicle, as detected with ultrasonography, can be used to predict ovulation
within a 24-hr period for most mares. Characteristics
of Ovulation A study was performed to determine ultrasonographic characteristics of ovulation (Carnevale, McKinnon, Squires, & Voss. 1988). Fifteen light-horse mares were assigned to the experiment upon acquiring the following preovulatory, follicular parameters: a) diameter of > 40 mm, b) marked softening upon palpation per rectum, c) pain upon palpation, and d) a change in shape from round to irregular. Preovulatory follicles were observed at < 1-hr intervals for 12 hr or continually when there were signs of impendi |