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Reproduction | Ultrasound

Ultrasound

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) Opportunity to re-examine the mare. In humans, no deleterious effects of ultrasonography have been reported. Frequent manipulation with the transducer within the mare's rectum or soundwaves at a frequency of 3 MHz have been determined not to be detrimental to continuing pregnancy (Selway. 1983; Vogelsang, Vogelsang, Lindsey, & Masset. 1989).

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 Kentucky during 1988 to 1989 (74 of  1211 foetuses) (Hong, Donahue, Giles, et al. 1993). Between 1986 and 1991 twins were associated with 221 of 3514 (6.3%)  aborted foetuses, still born foals or foals that died within 24 hours of birth (Giles, Donahue, Hong, et al. 1993). In cattle significant breed differences exist in ability to twin. Twinning increases from the first of fourth calf then plateaus. After the first twin calf is born, twinning is four to five times more likely compared to the normal population and in addition, within season variability affects twin numbers (Johansson, Lindhe, & Pirchner. 1974). Twinning in mares was responsible for 4% of dystocias (Leidl, Stolla, & Schmid. 1993). Between 1968 and 1981 in West Germany twin pregnancies averaged 3.6% of a total population of 13,710 pregnancies. Eighty five % ended in last trimester abortion, 5% as still born twins and 10% produced either one or two live foals. Pascoe (1983), recorded that of a 130 twin pregnant mares (rectal palpation) only 17 gave birth to life foals (13%) and furthermore only 38 of a 102 (37%) produced live foals the next year (Pascoe. 1983). A further study examining 1015 Standardbred and mares from 2 to 24 years of age, identified twin conception rate per cycle of 15.3%, 8.8% and 14% from maiden, lactating and barren mares respectively (p< 0.01) (Pascoe, Pascoe, & Wilson. 1987). In a study on abortion in Thoroughbred mares, twins accounted for 29% of an overall pregnancy loss of 12.8% (Platt. 1973). In a similar study, twins accounted for a still birth percentage of 40.9% compared to 1.2% for the single group. Of twins born live, 30% died in the first week compared to 1% of singles. Thus only 25% of twins that had made it to the last month of pregnancy survived the first 8 weeks of life (Platt. 1973). High rates of eCG have been detected in mares with twin pregnancy (Prohl, Busch, & Schuetzler. 1990) and these authors found twin pregnancy rates increased as the season progressed. The tendency for multiple ovulation and twinning to be familial was suggested by (Urwin & Allen. 1983). Although improved nutrition did not increase the rates of multiple ovulation (Woods, Scraba, & Ginther. 1982), it did appear to be related to a better display of oestrus receptivity. Vandeplashe (Vandeplassche. 1993) explained a high frequency of embryonic death and low vitality of twins by reduced blood supply associated with restricted uterine capacity in the later stages of pregnancy. He noted inter-placental vascular anastomoses were macroscopically visible in 25% of the cases which resulted in blood chimaerism that did not interfere with subsequent fertility. No freemartins were observed (Vandeplassche. 1993). More twins survive in draught mares and they have a similar incidence compared to Thoroughbreds (Vandeplassche, Podliachouk, & Beaud. 1970). Twins were more common as the season progresses and in younger mares (Jeffcott & Whitwell. 1973).

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 (³4 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 Griffin (1994) examined the natural outcome of bilateral twins (one in each horn) that were viable on day 40 in 15 pony mares. Readers should be aware that pony mares are not necessarily a good model for the Thoroughbred or other breeds as the incidence of twins is low and evidence is suggestive that the larger the breed (Draught, Thoroughbred and warmblood) the higher the probability of maintaining twins. Fifteen pony mares were monitored by ultrasonography until the outcome of the pregnancy was determined. Sixty six percent of the pregnancies resulted in either death of both (80%) or death of one (20%) during months two or three. Nothing occurred from then until month 8. Between months 8-11, two mares lost one foetus and two mares lost both. The two mares that lost one pregnancy both delivered undersized weak foals at birth. One mare (7%) delivered live twins at term and two normal foals were born from mares loosing the one pregnancy in month two. In this study six live foals were born (2 of normal size), from a total of 15 mares and 30 foetuses. This incidence is similar to previous reports wherein of 130 twin pregnant mares, only 17 live foals (13%) were produced (Pascoe. 1983). An interesting observation from the later report was that from the 102 mares that delivered live or dead twins in the previous year, only 37 produced live foals the next seasons and thus over two seasons there was an average of 23% producing live foals (Pascoe. 1983). An earlier study (Jeffcott & Whitwell. 1973), was extremely useful in categorising outcome of twins that managed to survive to later pregnancy. Twinning accounted for 22% of the cases of abortion and still-birth between 1967-1970. Sixty two sets of twins and their placentas were examined from Thoroughbred mares. All were considered to be dizygous. Abortion or still-birth of both twins from 3 months of gestation to term occurred in 64.5% of mares, although most (72.6%) slipped from 8 months to term. In the remaining cases one twin (21%) or both twins (14.5%) were born alive. Most foals at term were stunted and emaciated and of the 31 alive at birth only 18 had survived to 2 weeks of age (Jeffcott & Whitwell. 1973). In this study twin placentation was divided into three morphological groups according to the disposition of the chorionic sacks within the uterus. Type A placentation was seen in 79% of cases (48 sets of twins). One foetus occupied one horn and most of the body (mean 68% of the total functional surface area) while the other twin occupied only one horn and usually only a small part of the adjacent body. Where the chorions abutted there was a variable degree of invagination of the smaller chorion into the allantoic cavity of the larger twin. These pregnancies frequently ended in abortion or stillbirth of one or both twins. The larger twin had a much greater chance of survival than the smaller one. In this group 31/48 lost there pregnancies between 3 and 9 months (64.5%). The gestation length in this group was frequently shorter and at birth the larger twin had a much greater chance of survival than the smaller one. In this group only 6 foals of 48 sets were born alive. Of the 6 foetuses born alive 5 were the larger twin. Type B placentation occurred in 11% of cases (7 sets) and the placentas were orientated such that the villous surface areas were more or less equally divided and each foetus occupied one horn and half of the body. Both foals were usually similar in size and were usually born alive. Nine foals survived to 2 weeks from 6 sets of twins that made it to term. In this group (7 total) one aborted at 7 months. Type C placentation was seen in 10% of cases (6 sets of twins). In this group there was a greater disparity between the surface area of the 2 chorions. The smaller twin occupying only part of one horn, died earlier on and became mummified. The larger twin was usually born alive and a fair chance of survival. In this group 3 foals were born alive from 6 pregnancies at term (Jeffcott & Whitwell. 1973). The authors attributed the loss of twin foetuses and poor survival rates to placental insufficiency.

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 ( Cook , Australia ) is passed through the needle guide biopsy channel into either the heart , lungs or abdomen of the identified foetus. Penicillin is injected and the foetus monitored for the next 5 to 10 minutes. If the needle is in the chest or abdomen the demise of the foetus still occurs however it just takes a little longer. The apparent advantages of penicillin as we see them are: 1) it reduces iatrogenic bacterial contamination. 2) it can be visualised ultrasonographically as it is injected 3) foetal death can still be obtained without intracardiac needle placement. We have attempted to place mares on Regumate and long term oral antibiotics, however have found no difference in foetal survival rates with either treatment compared to those that have received no treatment. At the time of needle puncture mares are treated with systemic antibiotics for 3 days and intravenous phenylbutazone. Late in gestation twins maybe difficult to recognise with rectal ultrasonography except in those cases with observation of the twin membrane (Ginther & Griffin. 1994). Abdominal ultrasonography is useful to diagnose twins (Pipers & Adams-Brendemuehl. 1984; Rantanen & Kincaid. 1988). In the event of failure to diagnose twin pregnancies, occasionally abortion is heralded by lactation late in gestation (>7 months). There are reports of successful maintenance of pregnancy despite premature lactation (>1 month prior to foaling ) in cases with twin gestation. Apparently the premature lactation is induced by foetal death and the beginning of mummification of one foetus and thus is threatening to the remaining live foetus. In an initial report, 4 mares with apparent impending twin abortion were able to deliver live single foals concurrent with a mummified twin after supplementation with progesterone was initiated upon recognition of  inappropriate lactation late in gestation (Roberts & Myhre. 1983; Shideler. 1987). In these cases although foals were born small they survived and thrived normally. Further work will be necessary to determine which mares will respond best or even at all to supplementation with progesterone for initiation of premature lactation.

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 Wisconsin have suggested that in aged subfertile mares the primary problem may actually be the oocyte (Carnevale & Ginther. 1995). In the future, much more clear delineation of the various incidence of problems from the different parts of the reproductive tract will be forthcoming. The incidence of EED has been reported to be between 5 and 30% of established pregnancies (Day. 1940; Kenney. 1978). In recent studies, utilising ultrasonography, it appeared that EED occurred in mares much earlier than previously reported (Villahoz, Squires, Voss, & Shideler. 1985; Ginther, Bergfelt, Leith, & Scraba. 1985). Various causes and factors responsible for EED in mares, apart from presence of twins, have been suggested; which included nutrition (Belonje & van Niekerk. 1975; van Niekerk & Morgenthal. 1982), plant oestrogens and photo-period (Blythe & Kitchell. 1982), seminal treatments (Moberg. 1975), lactation stress, and foal-heat breeding (Merkt & Gunzel. 1979), genital infections (Day. 1940; Kenney. 1978; Merkt & Gunzel. 1979), chromosomal abnormalities, hormonal deficiencies (van Niekerk & Morgenthal. 1982), anabolic steroids (Villahoz, Squires, Voss, & Shideler. 1985), stress (van Niekerk & Morgenthal. 1982), failure of maternal recognition and deficiency of pregnant mare serum gonadotropin (PMSG) production.(Allen. 1984), immunological factors (Shivers & Liu. 1982; Liu & Shivers. 1982) and even a higher incidence from some individual stallions (Platt. 1973). Migration of the conceptus was originally believed to be a contributing factor in early embryonic death, however, it is now known to be a normal characteristic of the horse conceptus (Ginther. 1983). Lactating mares and mares bred during foal heat have been reported to have a higher incidence of EED than non-lactating mares (Merkt & Gunzel. 1979; Fiolka, Kuller, & Lender. 1985; Lieux. 1980). However, in a survey of 2,562 pregnancies in lactating and non-lactating mares, the incidence of EED was similar (Bain. 1969). Prior to the advent of ultrasonography, recognition and timing of EED was difficult. From data collected at CSU (Villahoz, Squires, Voss, & Shideler. 1985) over two breeding seasons involving 356 mares diagnosed pregnant utilising ultrasonography, the overall incidence of EED through day 50 post-ovulation was 17.3%. The majority (77.1%) of EED occurred prior to day 35 post-ovulation. During the period 15 to 35 days post-ovulation, a greater (P < 0.05) incidence of EED occurred between days 15 to 20 (26.2%) and 30 to 35 (29.5%) post-ovulation compared to other time periods. Maternal recognition of pregnancy has been reported to occur between 14 and 16 days post-ovulation (McDowell, Sharp, Grubaugh, Thatcher, & Wilcox. 1988; McKinnon & Squires. 1988). In the study at CSU, 13.3% of mares pregnant at day 15 lost their pregnancy by day 35. It should be noted that formation of endometrial cups occurs on approximately day 35.

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 impending ovulation. Ovulation was defined as a rapid decrease in follicular size characterised by disappearance of the large, fluid-filled, non-echogenic structure. A real-time, B-mode linear array scanner with a 5 MHz transducer was used for ultrasonographic examinations. Thirteen of 15 mares ovulated within the 12-hr examination period (mean = 85 min; range 15 min to 3 hr 37 min after beginning of observation). As ovulation approached, flattened or irregular images of the follicles were noted, concomitant with reduced follicular tone. This was likely due to diminished tensile strength of the follicular wall or perhaps a slow release of fluid from the follicle, although no fluid was visualised outside the follicle as ovulation approached. An echogenic nodule, approximately 5 to 10 mm, was noted within the follicles of two mares prior to ovulation These may have represented the cumulus oophorous, which has previously been visualised in women. Prior to ovulation, 10 of 13 follicles developed a tear in the follicular wall, which was characterised by a jagged protrusion of the follicular border toward the ovulation fossa. In seven mares, the tear or point was first observed an average of 41 min (range 15 to 77) prior to ovulation and was a consistent indicator of impending ovulation. A tear or pointed appearance, observed with ultrasonography just prior to ovulation, is likely due to breakdown of ovarian stroma and protrusion of the follicle toward the ovulation fossa. These observations probably parallel deterioration of the follicular wall, stigma formation and protrusion of the basement membrane just prior to ovulation, as observed in other species. Ovulation, defined as a rapid decrease in follicular size, occurred in an average of 42 sec (range 5 to 90). Little or no follicular fluid remained in the follicle after ovulation. Two mares failed to ovulate within 12 hr after initiation of scanning and subsequently formed anovulatory haemorrhagic follicles (AHF). This occurrence was possibly due to season (McKinnon, Squires, & Voss. 1987). Abnormal ovulations such as AHF’s and luteinised, un-ruptured follicles (McKinnon, Squires, & Pickett. 1988) may initially display a similar sequence of events as normal, preovulatory follicles without ovulation. This was characteristic of two AHF’s in the present study. Increased echogenicity of the follicular wall was visualised in all follicles prior to ovulation. Appearance of echogenic "spots" within the follicular fluid, probably due to dispersal of granulosa cells, was noted in 7 of 13 follicles (54%). However, echogenic "spots" within follicular fluid, or a bright, echogenic follicular border, were not consistently useful in predicting time of ovulation. A bright echogenic border, irregular shape and a tear in the follicular wall was predictive of imminent ovulation.

Efficacy of Ultrasonography for Determining Ovulation

In one study conducted, the accuracy of rectal palpation and ultrasonography for detection of ovulation was compared (Squires, Voss, Villahoz, & Shideler. 1983). Thirty-four normally cycling, non­lactating mares of light-horse breeds were used. Data collection began on day 2 of oestrus or when a > 35 mm follicle was detected by palpation. The mares were palpated rectally and scanned every 12 hours. Rectal palpation was performed by one investigator and ultrasonographic scanning by a second, each unaware of the diagnosis made by the other. A 3 MHz, linear array, real-time ultrasonographic scanner was used in the study. Mares that ovulated, based on palpation, but which had a second ovulatory-sized follicle continued to be palpated until a second ovulation occurred or the mare returned to oestrus. The technician utilising rectal palpation defined ovulation as absence of the follicle and a soft, sometimes painful indentation. Ovulation based on ultrasonography was defined as a change in the echogenic pattern characterised by disappearance of the large, black, fluid-filled, non-echogenic structure and the presence of an echogenic area. In 24 of 34 mares, ovulation was detected at the same time by both methods. Three ovulations were detected by ultrasonography 12 hr prior to detection by palpation and five ovulations were detected with ultrasonography 12 hr after detection by palpation. Thus, within ±12 hr, ovulation was detected by both methods in 32 of 34 mares. In one mare, detection of ovulation by ultrasonography occurred 36 hr prior to detection by palpation. In addition, ovulation was detected by ultrasonography in one mare, but the technician failed to detect ovulation in this same mare by palpation. Double ovulations were detected by ultrasonography in 3 of 34 mares. These ovulations occurred on the same ovary within 12 hr. These double ovulations were not diagnosed by rectal palpation. It may be that close apposition of two follicles on one ovary was responsible for failure of the palpator to recognise both structures. The recognition of double ovulation is important to prevent twin pregnancies, and in an embryo transfer program for correct scheduling of recipient mares. Although ultrasonographic scanning of mares' ovaries should not be used as a replacement for rectal palpation, it is obvious there are occasions where this technique may be more accurate. Even with daily, manual ovarian examination, there are times when ovulation(s) cannot be accurately determined. Use of ultrasonography in these circumstances allows one to make a more accurate determination concerning ovulation. In cases where ovulation is detected by ultrasonography and not by palpation, it is most commonly associated with a well-circumscribed developing corpus haemorrhagicum which, although smaller, has palpation characteristics similar to that of a fluid-filled follicle. These structures either collapse and refill with blood or apparently fail to collapse completely.

FORMATION AND DEVELOPMENT OF THE CORPUS LUTEUM

The CL is present during two-thirds of the mare's oestrous cycle, and for the first 6 months of pregnancy. Progesterone, a primary hormonal product from the CL, has a multitude of functions, including initiation and maintenance of pregnancy. Therefore, methods to evaluate the CL are extremely important. Because of the position of the CL within the ovary, palpation per rectum is of little value for identification and evaluation.  However, ultrasonography has been shown to be an effective and accurate means of identifying this structure.  Some of the reasons for ultrasonographic evaluation of corpora lutea are: 1) detection of ovulation; 2) evaluate CL formation; 3) determine size and characteristics of the CL; 4) determine if failure of a mare to display oestrus is due to prolonged maintenance of a CL or absence of a CL and follicular activity; 5) distinguish between anovulatory haemorrhagic follicles, luteinised un-ruptured follicles or CL; and 6) determine if a mare has ovulated more than one follicle. After rupture of the follicle, a corpus haemorrhagicum is formed as a transient phenomenon in the development of the CL in the mare. However, it was demonstrated in an ultrasonographic study (Pierson & Ginther. 1985) that the equine luteal gland may involve two ultrasonically distinct luteal morphologies. Both types of luteal structures are uniformly echogenic on day 1. One type, classified as uniformly echogenic, is seen in approximately 50% of the CL’s and the percent of echogenicity remains constant for the duration of dioestrus. The other, classified as centrally non-echogenic (corpus haemorrhagicum) develops a non­echogenic center on day 0 or day 1. The percentage of CL’s considered echogenic was lowest on day 3, and increased linearly throughout dioestrus.  In a subsequent study (Townson & Ginther. 1989), the time required for accumulation of fluid and formation of central clots (non-echogenic areas) were studied by ultrasonography. Examinations were conducted at 15 min intervals for the first 2 hr after ovulation, again at 8 hr and thereafter at 12-hr intervals for 5 days. In 2 of 10 mares, a non-echogenic area did not develop within the luteal gland, and in one mare only a small central area (0.5 cm2) was detected at 20 and 32 hr, and not thereafter. In five mares, a non-echogenic central area developed within the luteal gland after expulsion of follicular fluid. Size of the non-echogenic area varied from 0.5 to 11.6 cm2 For those mares with central non-echogenic areas, echogenic lines within the central area were detected. These were attributed to clotting and fibrinisation of the contents. From the results of data collected at CSU (McKinnon, Squires, & Voss. 1987), it appeared that, when CL evaluations were made on days 5 to 7 post-ovulation, the number of centrally non-echogenic CL’s was lower (9.2%, n=192 cycles) than that reported previously (Pierson & Ginther. 1985). In addition, the incidence of at least one centrally non-echogenic CL increased with double ovulations (36%; n = 23 double ovulations). However, from the results of more recent data, a higher percentage of centrally non-echogenic CL’s was observed (McKinnon, Squires, & Pickett. 1988). This is dependent, to some extent, upon days post-ovulation. Comparative studies, on duration of dioestrus, concentrations of progesterone and fertility data have been conducted to determine that both morphologic types of CL’s are normal (Townson, Pierson, & Ginther. 1989). Ginther  reported on the accuracy of detecting a CL with ultrasonography. Location of the CL was established by daily palpation per rectum. Ultrasonographic examinations were done by another technician unaware of the site of ovulation. The ultrasonographer recorded location of the CL, or indicated that one was not found or that there was uncertainty about identification. The ultrasonographer was correct in 88% of his examinations conducted on days 0 to 14 post-ovulation. In the remaining 12%, the ultrasonographer recorded the locations as uncertain. In addition, in all 12 mares that were in oestrus, the location of the CL was recorded as uncertain. From these results it appeared that ultrasonography can be used to visualise a CL, even if the site of ovulation is unknown. Therefore, ultrasonography is an extremely valuable diagnostic tool for determining the presence or absence of the corpus luteum. The ultrasonographic image is affected by amount of blood within the corpus luteum. Blood is non-echogenic, whereas luteal cells are echogenic. Generally, luteinisation begins on the periphery of the structure and migrates medially. Normally, as the CL ages, blood is resorbed and a uniformly echogenic, luteal structure develops. Fibrin-like material can separate the blood clot into areas of dark, non-echogenic sections containing red blood cells, plasma and/(or) perhaps follicular fluid. Lighter areas may be indicative of fibrin strands or developing luteal tissue. Although the ultrasonographic properties of the mature CL are similar to ovarian stroma, a CL can be distinguished by its defined borders. Ginther found that the ultrasonographic texture of the luteal gland was characterised by an echo pattern indicative of loosely organised, well-vascularised tissue, whereas ovarian stroma generally yielded brighter echoes in a pattern representative of dense tissue. Also, the majority of CL’s had a distinct mushroom or gourd shape. In glands classified as centrally non-echogenic, the non-echogenic area was first visible on day 0 or 1 post-ovulation. These types of luteal structures were at their greatest echogenicity on the day of ovulation (75 to 100% of the gland). This probably was due to the ultrasonographic properties of collapsed follicular walls. The non-echogenic area, which was the central cavity, enlarged over days 1 to 3 due to enlargement of the blood clot. As the blood clot resorbed, that portion of the structure that was echogenic increased throughout the remaining portion of the cycle. In contrast, luteal glands that were characterised as uniformly echogenic did not change throughout the cycle, except the brightness (grey scale) changed throughout the life of the corpus luteum. Ginther also demonstrated that both types of glands change in echogenicity throughout the dioestrous period. Initially, the CL is highly echogenic on the day of ovulation. At this time it is easiest to identify. The echogenicity decreases over the first 6 days of dioestrus, remains at a minimum level for several days during the middle of dioestrus, then increases over days 12 to 16. The very bright hyperechogenic echoes on day 0 may be due to apposition of collapsed follicular walls. There was also an increase in brightness of the CL during the time of CL regression. These ultrasonographic changes are apparently indicative of changes in luteal haemodynamics and changes in tissue density. With experience, the practitioner can become very accurate at using ultrasonography to confirm ovulation and to detect the presence of a corpus luteum. Ultrasonography can also be used to diagnose pseudo-pregnant mares. A persistent CL and absence of an embryonic vesicle are evidence of a pseudo-pregnant mare. Once these mares are identified, then prostaglandins can be safely given to induce oestrus. Echogenicity of this structure can be used to determine to some extent the age of corpus luteum. Hyperechogenicity is typical of the first few days after ovulation or during CL regression. The first few days usually can be distinguished from the last few days on the basis of gland size. In the middle of dioestrus, the CL will be lower on the grey scale than either at the beginning or the end. However, the structure will be at maximal size during the middle of dioestrus. If the CL contains a central non-echogenic cavity, the ratio of luteal tissue to blood clot and degree of organisation of the clot can be of assistance in estimating age of the gland. The blood clot develops during the first few days, then progressively becomes more organised and proportionally smaller.

OVARIAN ABNORMALITIES

The ability to non-invasively examine the mare's ovaries permits diagnosis of various forms of ovarian abnormalities and pathology. Some ovarian abnormalities that have been recognised with ultrasonography are: 1) multiple preovulatory follicles, 2) anovulatory haemorrhagic follicles, 3) luteinised, un-ruptured follicles, 4) persistent CL’s, and 5) various ovarian tumours and peri-ovarian cysts.

Multiple Preovulatory Follicles

Since the mare normally ovulates only one follicle during each oestrous cycle, multiple ovulations may be considered an abnormality. Breed influences the incidence of multiple ovulation. For example, Thoroughbreds, warm bloods and draft mares have been shown to have the highest incidence of multiple ovulation; whereas, Quarter Horses, Appaloosas and ponies have the lowest incidence with Standardbreds being intermittent (Ginther. 1982). Multiple, preovulatory follicles or ovulations may be particularly difficult to detect by rectal examination, especially when they are in close apposition on one ovary. In one study, more embryos were obtained from multiple ovulating mares that bilaterally ovulated than from those in which multiple ovulations were unilateral (Squires, McKinnon, Carnevale, Morris, & Nett. 1987). Multiple ovulations should be encouraged when ultrasonography is available to eliminate one of two developing vesicles at 14 days; because multiple ovulations increases the probability of conception. The ability to collect and transfer multiple embryos from a donor mare has the potential of improving efficiency of an equine embryo transfer program. The viability of embryos collected from naturally and induced multiply ovulating versus singly, naturally ovulating mares is similar (Squires, McKinnon, Carnevale, Morris, & Nett. 1987). Recovery of embryos from singly, ovulating mares was 53% compared to 106% for naturally doubly-ovulating mares. Pregnancy rates, 50 days after surgical transfer, were 68 and 129%, respectively. Treatment of normally, singly ovulating mares with equine pituitary extract resulted in two embryos recovered per donor compared to 0.65 for control (Squires, McKinnon, Carnevale, Morris, & Nett. 1987). Non-surgical pregnancy rates for embryos collected from superovulated mares were identical to those obtained for untreated controls.

Anovulatory Haemorrhagic Follicles

Anovulatory haemorrhagic follicles (AHF) are the result of preovulatory follicles growing to an unusually large size (70 to 100 mm), failing to ovulate, then filling with blood and gradually receding. Ultrasonography has been used by us to confirm this condition in mares when it was first identified as an abnormality by rectal palpation. This phenomenon may be recognised as an entity distinct from a corpus haemorrhagicum by its size and by ultrasonographic characteristics. The blood in an AHF is distinctly echogenic, whereas normal development of the corpus haemorrhagicum results in a generally non-echogenic central blood clot (15 to 35 mm) in diameter.  However, both may have criss-crossing fibrin-like strands. The formation of luteal tissue around the periphery of an AHF follicle is rare or minimal. We have noted in some mares, development and subsequent ovulation during the same oestrous cycle of another follicle after formation of an anovulatory haemorrhagic follicle. In these mares, behavioural signs of oestrus persisted throughout an unusually long cycle of approximately 12 days, or 5 days after recognition of an anovulatory haemorrhagic follicle.  Unfortunately, serum progesterone has not been measured in these animals. It is possible that AHF's are the previously reported "autumn" follicles, since most have occurred toward the end of the ovulatory season. Perhaps AHF’s develop because of insufficient stimulus for ovulation from gonadotropic releasing hormones. After the last ovulation of the year, mares may develop a large follicle at the expected time, but the follicle does not ovulate and the mare enters the anovulatory season.

Luteinised Un-ruptured Follicles

Although anovulatory oestrous periods are common during the anovulatory season, they are rare during the ovulatory season. An incidence of 3.1% was reported in Thoroughbreds and Quarter Horses (Hughes, Stabenfeldt, & Evans. 1972), and even these may have been misdiagnosed because palpation was used. Luteinised, un-ruptured follicles have been reported in women and mice, but not in non-pregnant mares. This phenomenon is thought to be associated with reproductive senility. In one study that was initiated to recover embryos from oviducts of old, infertile mares, on some occasions when the oviducts were flushed 2 days post-ovulation, no embryos or unfertilised ova were recovered and from close examination of the ovulation fossa it appeared that recent ovulation had not occurred (McKinnon, Squires, & Pickett. 1988). Surgical removal of two ovaries from two mares confirmed that ovulation into the ovulation fossa had not occurred, and from prior ultrasonographic examination it appeared that an atypical corpus haemorrhagicum had formed. Both mares ceased displaying signs of oestrus within 1 day of the suspected ovulation. Concentrations of progesterone levels were not available. These structures may have been luteinised, un-ruptured follicles similar to those in women and mice and may be associated with senility. Luteinisation without ovulation occurs quite commonly in pregnant mares in association with formation of secondary CL’s (Squires, Douglas, Steffenhagen, & Ginther. 1974).

Prolonged Maintenance of the Corpus Luteum

Rectal palpation of the CL, although possible on occasion, is generally unrewarding. Prolonged maintenance of the CL, resulting in pseudo-pregnancy can be differentiated from an anovulatory or anoestrous condition by ultrasonography. The CL is first visible on day of ovulation (day 0) as a strongly echogenic, circumscribed mass of tissue (Pierson & Ginther. 1985). The echogenicity gradually decreases throughout dioestrus. However, just prior to regression of the CL, echogenicity increases. This may reflect changes in luteal haemodynamics. In one study (Pierson & Ginther. 1985), the CL could be observed for a mean of 17 days (n = 55). On occasion, the presence of a CL may be seen as a circumscribed, highly echogenic area of tissue in the ovary, in mares that failed to return to oestrus at the expected time. Prolonged maintenance of the CL is more commonly recognised in normally cycling mares that have been bred. Generally, the mare fails to return to oestrus at the expected time, even though she is not pregnant. Perhaps pregnancy is initiated and the embryo prevents secretion of prostaglandin F2-alpha prior to undergoing early embryonic death. In one study (McKinnon & Squires. 1988), removal of the conceptus early in pregnancy (days 7 to 11) resulted in return to oestrus at the expected time or slightly earlier, while removal later (days 14 to 16) resulted in prolonged maintenance of the CL, or pseudo-pregnancy. In our practice ultrasonography became more commonly accepted due to our abilities to diagnose retained CL’s and predict the mares response to PGF2µ. These stage of cycle exams are now routinely performed on all mares arriving at stud late in the breeding season and on any mares not showing heat within a three week period of arriving.

Ovarian Neoplasia

The incidence of ovarian tumours is relatively common in mares when compared to other domestic species.  The incidence of ovarian tumours in horses has been reported to be as high as 5.6% of all neoplasms. By far the two most common tumours are granulosa thecal cell tumours and teratomas. Rarely, ovarian enlargements associated with a cystadenoma may be detected (Hinrichs, Frazer, deGannes, Richardson, & Kenney. 1989). Granulosa theca cell tumours are usually large, benign steroid-producing tumours often associated with behavioural changes and poor reproductive performance. The most common history is a barren, anoestrous mare. Other clinical signs are intermittent or continuous oestrus, nymphomania or stallion-like behaviour. The ultrasonographic characteristics of granulosa thecal cell tumours will vary. Gross characteristics may be solid or cystic,. Palpation may reveal a smooth surface, a knobby, hard surface, or sometimes a soft surface with obvious follicular development. The unaffected ovary is usually small and inactive, however on occasion mares with a cycling contralateral ovary may be detected (Hinrichs, Watson, & Kenney. 1990). Surgical excision is the treatment of choice, and most mares will return to normal reproductive performance within 2 to 16 months after surgery. Ovarian teratomas are benign and non-secretory. The tumours arise from germ cells and are usually nondescript, epithelial tissue, but may contain cartilage, skin, bone, hair, nerves, sebaceous material and even teeth. They may be solid or cystic. They generally do not interfere with fertility and are most commonly discovered during routine rectal palpation, unless they become extremely large and affect other organs. Ultrasonographic examination may help differentiate between neoplasia and other large non-neoplastic structures, such as anovulatory haemorrhagic follicles or an ovary during the transitional period with multiple, non-dominant follicles. However, in general, definite diagnosis will rely on histological or gross examination of the affected ovary. Recently attention has focused on inhibin levels (Piquette, Kenney, Sertich, Yamoto, & Hsueh. 1990; McCue. 1992) for diagnosis. In one study elevated inhibin was accurate in 87% of cases compared to 54% of positive diagnoses made with elevated testosterone (McCue. 1992). At the GVEH we routinely use inhibin levels to assist with the diagnosis of granulosa theca cell tumours. Samples are sent to Monash University and results of > 1 ng/ml are suspicious.

Peri-ovarian Cysts

Embryonic vestiges and cystic accessory structures associated with the ovary and oviduct are quite common in mares. These cysts, although often small, may occasionally be confused with an ovarian follicle. Rectal palpation in these circumstances is generally more accurate than ultrasonography in determining whether the structure is part of the ovary. Small fimbrial cysts (< 10 mm) probably do not cause infertility, however on occasion, cystic remnants of the mesonephric tubules and ducts may grow quite large (30 to 40 mm in length and 10 to 15 mm in diameter). One such recognised cyst is the Hydatid of Morgagni. This type of cyst has been diagnosed with ultrasonography, although it is sometimes difficult to distinguish between ovarian follicles and peri-ovarian cysts.

Miscellaneous Ovarian Abnormalities

Hydrosalpinx is not common in mares, but since it is a fluid-filled structure, it may be detected with ultrasonography. Definitive diagnosis will probably require laparoscopy or exploratory surgery. Information on other types of ovarian abnormalities is just beginning to be obtained. We have identified cystic, follicular structures that have not ovulated. Some have regressed while others have persisted. Only careful documentation, hormonal and histological analyses will determine the aetiology of and treatment for many of these previously unidentified abnormalities.

The Stallion

Ultrasonography of the stallion is only just beginning to become recognised as a source of good diagnostic information. Listed below are some conditions we routinely use ultrasonography to diagnose. We feel ultrasonography has become an almost indispensable diagnostic tool.

Internal reproductive tract

Accessory sex gland and internal ring palpation are occasionally part of a stallion fertility examination. While the incidence of recognised accessory sex gland abnormalities are low (Little & Holyoak. 1992), they are not always able to be diagnosed or even suspected from the history, a physical exam or even semen evaluation. Stallions with high numbers of leucocytes or other abnormal cells in their ejaculates are good candidates for transrectal ultrasonography. This should be performed in conjunction with culture and cytology of semen, endoscopy and palpation. A correlation between sexual development, hormonal environment and accessory sex gland development has been demonstrated (Holyoak, Little, Vernon, McCollum, & Timoney. 1994). Structures able to be identified by transrectal ultrasonography are the prostate gland, ampullae of the deferent ducts, vesicular glands, excretory ducts (all meet at the seminal colliculus), pelvic urethra, bulbourethral glands and abdominally located testicles.

Ampullae

The ampullae are enlarged, glandular portions of the terminal vas deferens (deferent ducts). They are readily located as they  converge over the neck of the bladder and pass under the prostatic isthmus. They are well recognised as tubular structures that may have a non echogenic centre, particulary in aroused stallions. Transrectal ultrasonography should be performed in cases of suspected uni or bilateral excurrent duct obstruction. Blockage most commonly occurs in the ampulla and may occasionally be felt rectally. Ultrasonographically the blockage can be seen as a hyperechoic mass in the ampullae. A 7.5 MHz probe is better for fine detail of smaller structures such as the ampullae and seminal vesicles (Little & Woods. 1987; Little & Holyoak. 1992). At the GVEH we have been referred numerous cases of ampullary plugging or blockage. The common history is that the stallion has had normal fertility and at the begging of the subsequent breeding season does not get any mares (or very few) mares in foal. Seminal evaluation reveals markedly reduced sperm numbers compared to the stallions testicular size. Quite often none are identified at all and yet the testicles are of good consistency. Repeated administration of oxytocin followed by ejaculation 30 min to 1 hour later in most cases will clear the obstruction. Some refractory cases have needed tranquillisation with xylazine and rectal massage as well. When these stallions have been sexually stimulated prior to xylazine administration, many stallions will spontaneously ejaculate, most within 2 minutes (McDonnell & Love. 1991). These cases frequently re-occur.

Ejaculation can be visualised transrectally (Weber & Woods. 1991) and this technique has been able to demonstrate that the ampullae dilate rhythmically prior, during and even after the ejaculation (Weber & Woods. 1993).

Vesicular glands

The vesicular gland (seminal vesicles) are pyriform sacs that produce and store the gel fraction of an ejaculate. The size and echotexture of the vesicular glands depend on the degree of sexual stimulation. Teasing dramatically increases the amount of gel produced (Pickett, Amann, McKinnon, Squires, & Voss. 1989).The incidence of seminal vesiculitis problems is much lower than bulls, however we have identified some stallions with bacterial adenitis. In these cases the gel portion of the ejaculate commonly has had large amounts of necrotic debris and even blood clots. We have seen this associated with haemospermia (McKinnon, Voss, Trotter, Pickett, Shideler, & Squires. 1988). Occasionally rectal palpation does not demonstrate pain (Blanchard, Varner, Hurtgen, et al. 1988) and laboratory back up (white blood cells), fractionation of semen, expressed secretions and even endoscopy of the vesicular gland openings are indicated to support the diagnosis. Recently ultrasonography has been useful in diagnosis (Malmgren & Sussemilch. 1992; Malmgren. 1992; Freestone, Paccamonti, Eilts, McClure, Swiderski, & Causey. 1993). In these cases ultrasonographic findings were increase in overall size, thickened wall and increased echogenicity when compared to the contralateral unaffected gland. In one case colic was the presenting clinical sign and the stallion had discomfort on direct palpation of the seminal vesicle and when sexually aroused (Freestone, Paccamonti, Eilts, McClure, Swiderski, & Causey. 1993). Treatment is sometimes difficult and refractory cases are not uncommon. Presumably this is due to poor penetration of antimicrobials to the gland tissue (Blanchard, Varner, Love, Hurtgen, Cummings, & Kenney. 1987). Long term antibiotic administration (Trimethoprim-sulfamethoxazole) has been successful, however on occasion more aggressive therapy must be considered such as direct lavage through an endoscope or even surgical removal (Klug, Deegen, Martin, Bader, Lieske, & Freytag. 1979; Varner, Schumacher, Blanchard, & Johnson. 1991). In some refractory cases, treatment of the semen with antibiotic laden extenders (Blanchard, Varner, Love, Hurtgen, Cummings, & Kenney. 1987) and breeding after 30 min to 1 hour after mixing will prevent bacterial contamination (Pickett, Squires, & McKinnon. 1987).

Pelvic urethra

The pelvic urethra runs from the neck of the bladder to the ischial arch. The urethra is difficult to see and may only be evident by specular reflection. There is a wider dilation to the urethra next to the colliculus that becomes visible ultrasonographically as the stallion becomes sexually aroused and releases pre-ejaculatory fluid. Few problems are visualised at the pelvic urethra apart from rare cases of rents in the urethra that communicate with the stratum cavernosum.

Inguinal rings

At the GVEH, internal reproductive ultrasonography is always (mostly) performed in cases of suspected cryptorchidism. It is quite easy to identify an abdominally retained testes (Jann & Rains. 1990) once the operator understands the normal echotexture of the testicle. The echotexture is reduced in density slightly compared to the external testis. In our experience no case of abdominally located testis has been mis-diagnosed, however occasionally an extra-abdominal testes will not be identified in horses with excessive peri inguinal fat. The advantages of pre-surgical identification of the testes are to enable accurate scheduling of the following surgery (more quickly) if the testes is extra-abdominal, a definitive pre-surgical placement of the testicle provides confidence to enter the abdomen if difficulty is encountered during surgery and on occasion we have identified large neoplastic testicles in the abdominally retained testes. In one case of bilateral cryptorchidism intra-operative transrectal ultrasonography was used to actually guide the surgeons hand to the testicles through a dilated inguinal ring approach. Multiple cases of male pseudohermaphrodites have been identified at the GVEH, and in one instance a familial distribution was suspected with three siblings affected (JE Axon pers comm). In all cases of male pseudohermaphrodites we have seen, the classical echotexture of testicular tissue was useful in confirming the diagnosis. Male pseudohermaphrodites externally are females (64 XX) but have testicles that are almost exclusively abdominal cryptorchids. Behaviour varies but mostly is stallion like. The vulva is often displaced ventrally with varying degrees of enlargement of the clitoris to one resembling a short penis. Occasionaly chronic anabolic steroid administration may cause failure to cycle, male behaviour and an enlarged clitoris. These cases should be able to differentiated using ultrasonography.

External reproductive tract

External reproductive tract examination includes the scrotal contents (spermatic cords, testes, epididymides, vaginal cavities and scrotum) and the external penis. Commonly a 5 MHz scan head is used however a 7.5 or even a 10 MHz would be preferable for many of the structures visualised. Due to the irritant nature of some lubricants special care of the sensitive scrotal skin is advised. We commonly use mineral oil and then later wash with a mild soap or detergent. The optimal time to evaluate most stallions is within a few minutes of ejaculation, which is when most stallions are relaxed.

Scrotal contents

Vaginal cavity

An enlarged scrotum is a diagnostic challenge for the clinician. The three areas that generally can enlarge are the testes, the vaginal cavity or the surrounding layer of the skin, tunic and fascial layer. Ultrasonography is particularly useful in differentiating these enlargements. In general the skin and vaginal cavity are usually the areas of enlargement, probably because they are easiest for fluid to accumulate in compared to the testicle that has a relatively thick, non-elastic tunic. Fluid that accumulates between the testes and the parietal vaginal tunic (vaginal cavity) is termed a hydrocoele if it is clear, a haematocoele if it contains blood and a pyocoele if it contains many white blood cells. Hydrocoeles are the most common fluid accumulation (Varner, Schumacher, Blanchard, & Johnson. 1991). The epididymis, due to its ventral location and enlargement of the tail, is the area that is most commonly surrounded by fluid. This fluid often outlines the tail. Hydrocoeles are often transient and often reflect non reproductive pathology such as low protein, abdominal neoplasia and even on occasion chronic peritonitis with increased abdominal fluid. Haematocoels are generally associated with trauma and are recognised by fibrin strands and even swirling non clotted blood. The visceral tunic should be evaluated for integrity if a haematocoele is present and testicular removal contemplated if a rent is demonstrated. Pyocoeles are less common and are seen with orchitis, peritonitis, secondarily infected hydrocoeles or haematocoeles and on occasion after a penetrating wound. Scrotal hernias (inguinal hernias, ruptured inguinal hernias or inguinal ruptures) can also be diagnosed with ultrasonography.

Scrotal skin and investments

Oedema secondarily to trauma, allergic reactions, dependant oedema and after application of irritants are causes of scrotal skin thickening. Ultrasonography may be necessary to differentiate between firm scrotal oedema and a haematocoele, although many of these conditions may occur concurrently.

Spermatic cord

The spermatic cord can twist within the scrotum if there is enough movement allowed by the caudal ligament of the epididymis or proper ligament of the testes. We assume most spermatic cord torsion is intra-vaginal (excluding the parietal layer of the vaginal tunic) although the relative frequencies of intra versus extra-vaginal cord torsions have not been reported for horses (Varner, Schumacher, Blanchard, & Johnson. 1991). In addition the term testicular torsion ,although more commonly used, does not accurately describe the condition as it is the spermatic cords that has twisted. It is simple to diagnose a 1800 degree torsion by location of the tail of the epididymis cranially. Our belief is that any stallion can develop a minor 1800 torsion that can spontaneously correct, but that the older stallion is more likely to develop permanent 1800 torsion due to stabilisation of the testes by adhesions between the vaginal tunics that are presumably associated with strongylus edentatus migration (Smith. 1973; Pickett, Amann, McKinnon, Squires, & Voss. 1989). Spermatic cord torsion of 1800 has been associated with slightly reduced spermatozoal production (Pickett, Voss, Bowen, Squires, & McKinnon. 1987). No attempt is made to correct a 1800 torsion. Torsion of the spermatic cord of greater than 1800 results in much more serious complications (Varner, Schumacher, Blanchard, & Johnson. 1991). Torsions of 3600 or greater are treated as emergencies. Commonly these are associated with thickening of the cord, oedema and painful enlargement of the scrotal contents. The primary differential diagnosis for spermatic cord torsion is inguinal/scrotal herniation. An oedematous internal vaginal ring may occur with both conditions and ultrasonography is useful in the differentiation. Other conditions that may mimic torsion are scrotal trauma, periorchitis, neoplasia, haematocoele, varicocoele and thrombosis of the spermatic cord vasculature (Horney & Barker. 1975).

Another condition affecting the spermatic cords is the varicocoele. A varicocoele forms from abnormally distended and tortuous veins of the pampiniform plexus. The condition is well recognised in humans and rams. Their relationship to fertility has not been adequately studied in stallions. The source of infertility associated with varicocoeles in men is disturbance of the thermoregulatory mechanisms. In sheep breeding from affected rams is discouraged because of a possible inherited basis. In men, the condition may respond to surgery or more commonly thermo-regulation with long term commitment to water jacketed cooled underpants.

Testicle

There are many conditions of the testicles that can be diagnosed or detected with ultrasonography. Cryptorchidism has been discussed above. Testicular neoplasia, although uncommon may occur in any area of the testicle and can be visualised quite well due to different echogenicity. Germinal testicular tumours (originating from germ cells of the seminiferous epithelium) are in decreasing order of frequency: seminoma, teratoma, teratocarcinoma and embryonic carcinoma (Varner, Schumacher, Blanchard, & Johnson. 1991). Somatic tumours (non germinal derivation) are classified as parenchymatous such as Leydig and Sertoli-cell tumours and non-parenchymatous such as lipomas, fibromas and leiomyomas (Varner, Schumacher, Blanchard, & Johnson. 1991). Most equine testicular tumours are unilateral and of germinal derivation. Seminomas accounted for 87% in one large survey (Reifinger. 1988). All are treated as potentially malignant. Tumours are more common in retained abdominal testes (Hunt, Hay, Collatos, & Welles. 1990; Smith, Morton, Watkins, Taylor, & Storts. 1989; Gelberg & McEntee. 1987; Parks, Wyn-Jones, Cox, & Newsholme. 1986; Stick. 1980; Smyth. 1979). In horses with testicular tumours growth is usually reported to be slow. The first sign is often a harder and slightly enlarged testicle that is discovered on routine examination such as insurance etc. Intra scrotal masses may be identified as intra or extra testicular and may be solid or cystic. Testicle tumours frequently have a decreased echogenicity, however this depends on the amount of fluid within the tissue, the tissue type and growth rate. Prompt orchidectomy is the only treatment. Prior to removal of the affected testicle, ultrasonography of the contralateral testicle and an internal reproductive tract examination must be performed to detect malignancy etc. Semen evaluation will often remain almost unaffected early in the course of the disease. Following castration some testicular compensatory hypertrophy may occur, especially in young stallions (Hoagland, Mannen, Dinger, et al. 1986; Hoagland, Ott, Dinger, et al. 1986). Unilateral castration of stallions was associated with significant increase in serum LH and FSH concentrations and, perhaps, higher intratesticular testosterone, which may explain, in part, the compensatory hypertrophy noted in the remaining testis (Hoagland, Mannen, Dinger, et al. 1986).

Testicular trauma may result in a testicular haematoma or chronic testicular degeneration. Testicular haematomas are discrete and rarely cause a rent in the visceral tunic. They are quite frequently painful on palpation and the scrotal contents enlarged. Because they are mostly traumatic in origin other signs are present as well, such as scrotal oedema. Testicular degeneration may be recognisable on palpation by a smaller and softer feeling testicle than the contralateral one. Ultrasonographically there is often an overall increase in echogenicity to the affected testicle, a reduction in size and a reduction in circulation to the testicular parenchyma as exemplified by loss of visibility of the central vein (Love. 1992). Arteries that penetrate the tunica albuginea may also appear more prominent at the periphery of the testis due to a reduction in blood flow to the testicular parenchyma. Orchitiscan be bacterial, viral, parasitic or autoimmune in origin. Bacterial orchitis may be associated with haematogenous spread of Strep. zooepidemicus, Strep. Equi, Actinobacillus equuli, Pseudomonas mallei, Salmonella abortus equi or Brucella abortus. Local invasion of the testes can be secondary to peritonitis or a penetrating wound and frequently involve Strep. spp, Staph. spp and E. coli (Varner, Schumacher, Blanchard, & Johnson. 1991). Retrograde infection through the epididymis is also possible. Either uni or bilateral orchitis can develop. The affected testicle(s) is hot and painful. The horse frequently has an elevated temperature. Intra and extra testicular oedema is present and frequently the horse will refuse to breed. If semen can be obtained then frequently spermatozoal motility is decreased, morphological abnormalities are increased and large numbers of leucocytes are present. On occasion orchitis may develop into a testicular abscess. At the GVEH we had a 7 yo stallion present with an enlarged scrotum and ultrasonography demonstrated an intra-testicular abscess with periorchitis as well. Unilateral open castration resulted in the horse returning to breeding and successfully impregnating mares 120 days after the surgery. Medical management of bacterial orchitis should include appropriate antibiotics, anti-inflammatory medication and even cold hydrotherapy. Viral orchitis is not as well understood and may be more important as a cause of testicular degeneration than we currently recognise. Equine influenza, viral arteritis and EIA have been implicated (Varner, Schumacher, Blanchard, & Johnson. 1991). Parasitic orchitis has been associated with the migration of Strongylus edentatus causing testicular irritation and adhesions between the tunics (Smith. 1973). Autoimmune orchitis may result from testicular trauma and damage to the blood-testis barrier. Spermatozoa are highly antigenic and it is postulated that developing spermatozoa are protected from the immune system by the blood testis barrier. Localised granulomatous reactions and degeneration of the testicle are the sequelae to extravasation of spermatozoa from lacerations, biopsies, neoplasia or trauma to the testicle. Anti-sperm antibodies have been documented for the stallion (Lee, Hunter, & Joo. 1995; Teuscher, Kenney, Cummings, & Catten. 1994; Nie, Lee, Momont, & Joo. 1993) and is a well recognised association with infertility in humans (Gupta & Garg. 1975).

In addition to examination for disease processes ultrasonography can used to measure the length, width and height as well as the cross sectional area and the circumference at the widest part of each testes. Using the formula for the volume of an ellipsoid (4/3 p abc, a = height/2, b = width/2, c = length/2) the volume of a testes can be measured. Semen of 26 stallions, 2-20 years of age, was collected once a day for 7 consecutive days to determine daily sperm output (DSO). After the last collection, 17 stallions were castrated. Testicular volume was estimated using two methods. Length, width, height, of each testis were measured by calliper. Length, width, height, cross-sectional area and circumference at the widest point of the testis were measured by ultrasonography. Both calliper and ultrasound measurements were first made in the live animal, and again in vitro. There were no differences in these measurements because of method or condition of the animal, ie., whether the measures were taken in the live animal or after castration. It was concluded that ultrasonography was a method of accurately estimating testicular volume (r = 0.99) as well as predicting DSO (r = 0.92) in the stallion. The authors of this report suggest that this estimation can be performed by substituting values derived by ultrasonography or callipers (Love, Garcia, Riera, & Kenney. 1991).

Epididymis

Conditions detected in the epididymis are cysts, inflammatory reactions and dilation of the tail lumen. Epididymal cysts are round and generally smooth. They are not very common and unless they become quite large do not interfere with fertility. The cysts most commonly are located in the head and body. Occasionally cysts form from extravasation of sperm in blind-ended efferent ductules in the caput epididymis. Infectious epididymitis is rarely seen as a primary entity and is most commonly associated with concurrent orchitis. The tail (cauda epididymis) is most commonly involved although the head (caput epididymis) and body may also be affected (Varner, Schumacher, Blanchard, & Johnson. 1991). Acute epididymitis can cause severe pain with swelling and oedema, that may later progress to abscessation, fibrosis and periorchitis. Ultrasonographically, the diameter of the head and tail of the epididymis is often larger than normal, hyperechoic in relation to the testis, and an irregular border is detected. (Held, Adair, McGavin, Adams, Toal, & Henton. 1990) Several bright 1- to 5-mm-diameter echogenic areas that alternated with less echogenic areas were seen in the head of the epididymis in one report (Traub-Dargatz, Trotter, Kaser-Hotz, et al. 1991). Ultrasonographic findings were interpreted as fibrosis attributable to chronic inflammation (Traub-Dargatz, Trotter, Kaser-Hotz, et al. 1991). Care in interpretation is needed as similar findings were also reported in a stallion with generalised lymphosarcoma (Held, Mccracken, Toal, & Latimer. 1992). Examination of the ejaculate reveals increased abnormalities and abundant white blood cells. Culture of the ejaculate may help identify the bacteria. Treatment of infectious epididymitis is difficult and even unilateral castration is often unrewarding due to the likelihood that the other epididymis is involved (Traub-Dargatz, Trotter, Kaser-Hotz, et al. 1991; Held, Adair, McGavin, Adams, Toal, & Henton. 1990). Epididymal sperm granulomas are seen with loss of spermatozoa from the excurrent duct lumen (Blue & McEntee. 1985). These are sequelae from a variety of causes such as, parasite migration, lacerations, trauma and epididymitis. Sperm granulomas may initially be painful but become indurated painless masses with time (Varner, Schumacher, Blanchard, & Johnson. 1991). Ultrasonographically these are quite hyperechoic. Spermiostasis is seen with an abnormal accumulation of sperm in the tail of the epididymis. This is associated with sexual rest and some form of dysfunction of the epididymis. Ejaculates from these stallions frequently have very high numbers of spermatozoa (30X109)and have a high percentage of detached heads and concurrent poor progressive motility. We call these stallions accumulators. Quite often the ampullae are affected as well. Seven to eight ejaculates (sometimes more) are necessary to return spermatozoal transit time through the epididymis back to normal. Ultrasonography may be usefull in determining abnormal gland storage.

Penis

Ultrasonographic diagnosis of penile trauma has been reported (Varner, Schumacher, Blanchard, & Johnson. 1991). Changes may be seen in several compartments after these insults. Oedema may be seen between the tunica albuginea and in the preputial membrane. Blood clots may be visualised in the corpus cavernosum (Hyland & Church. 1995). Tears in the corpus can be detected as well as vascular shunts. Clots associated with penothiazine induced priapism can be imaged and then later the fibrosis that accompanies long standing organisation of thrombosis (Varner, Schumacher, Blanchard, & Johnson. 1991).

Prepuce

Ultrasonographic diagnosis of preputial lesions is limited to space occupying problems such as posthitis (traumatic etc), oedema, preputial abscesses and neoplasia such as squamous cell carcinomas, papillomas, sarcoids and melanomas (Varner, Schumacher, Blanchard, & Johnson. 1991). On occasion it is useful to alert the clinician as to where the best place for drainage of an abscess or haematoma would be.

Conclusion

Ultrasonography has become indispensable to our practice. Fortunately much of the equipment used for reproductive ultrasonography can be used to examine many other parts of the horse. Ultrasonography is more fundamental than any other diagnostic technique. I would not want to practice veterinary medicine again without it.

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