Use of ultrasonic echography in equine gynecology

THERIOGENOLOGY USE OF ULTRASONIC ECHOGRAPHY IN EQUINE GYNECOLOGY (a) E. Palmer end M. A. Driancourt I.N.R.A. - ReproductivePhysiology 37380 Nouzilly (F.) Received

for

publication:

12/3/79

ABSTRACT

Ultrasonic echography of the reproductive tract of the nare was performed by introducing the multicrystal probe of a real time echoscope into the rectum of mares. This technique allowed for an improved estimate of ovarian follicular population when compared to rectal palpation. Visualization of the conceptus was possible from day 14 of pregnancy, giving a very accurate (292%) early pregnancy diagnosis. Photographs of ovaries and conceptus of different ages are presented, INTRODUCTION Ultrasonic echography is increasinglyused in human medicine for visualizing cyst-like structures without destroying them. Liquid phases do not exhibit any echo when exposed to ultrasounds, whereas the echoes emitted by tissues are strong. This observation suggested the possibility of visualizing ovarian follicles of the mare, as well as the young conceptus, as soon as it takes the form of a vesicle of sufficient size. MATERIALS

AND METHODS

. The apparatus used was an ALOKA SSD 202 real time -B- scope with a multicrystal probe with a frequency of

3.0 MHZ. . Animals were of various breeds of large horses (500 kg) or Welsh pony mares (250 kg). Ia) This work has been presented at the Annual Meeting of E.A.A.P. held in Harrogate (G.B.) 22-26 July 1979. ACKNOWLEDGEMENTS We wish to thank the AHS-France Company who provided the apparatus used in this study.

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. After having emptied the rectum by hand, as before rectal palpation of the genital tract, the probe was pushed into the rectum and directed towards the organs to be examined. Experiments have been performed to find the best approich to the ovaries of the mare, to describe the appearance of the ovaries, to estimate the accuracy of follicular measurements, and to present some physiological observations obtained with this technique, Visualization of the uterus and its contents during early pregnancy has been performed in our research station as well as in stud farms where mares were examined in the breeding stall. The apparatus was kept in a car placed behind the stall. Accuracy of pregnancy testing was measured by compaiing the results with later information such as return to oestrus, progesterone determination (l), PMSG tests and oestrogen determinations (2). RESULTS 1. - Visualization of ovaries by ultrasonic echography When the probe is directed towards the ovary, a section of the ovary consisting of the follicles is visualized on the screen (Photo 1). The liquid phases appear in black, whereas the surrounding tissues are white. The outlines of the ovary are more difficult to observe as they show no contrasts with the digestive tract in the abdominal cavity. In consequence, ovaries which bear no follicle of sufficient size (e7-8 mm) are difficult to visualize. Young corpora lutea (less than 5 days old) appear as high echo structure and present a very bright (lor 2 days old CL) or bright (3 to 5 days old) area (Photo 2). As the lateral resolution of the apparatus is about 5-10 mm, the picture shows a cross section of the ovary of similar thickness, and thus it is necessary to move the probe in order to visualize successive sections of the ovary to describe the whole follicular population, Different positions of the probe are necessary to be sure that adjacent follicles have been separated, since a wall between two vesicles gives strong ethos only when perpendicular to the ultrasonic waves. Horizontal sections are obtained with the probe rotated laterally in the rectum and maintained at its normal position. Vertical sections are obtained by pushing the rectum laterally under the ovary and directing the probe upwards. 2. - Validation of measurement of follicular size by echography A comparison of follicular measurement by rectal palpation or echography was made with reference to post-

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mortem sectioning of ovaries of 4 mares. The three largest follicles of each of the 8 ovaries studied were estimated independentlyby two different observers. Agreement of the measurementswas estimated by the correlation and regression of experimentalmeasurements to direct measurements of post-mortem sections, An ideal estimate should give equal mean measurements of follicular size, a correlation r*l end a slope of regression line r=l. Very good estimation of the largest follicle is obtained bv both valuation (r=0.92t b=0.72) and echocrraDhv (r=O.gli bkO.99) kith a slightly-betterregression f& the latter. For the 2nd and 3rd follicle, estimation of size is of much lower quality with both techniques. However, the superiority of echography vs. palpation is clear with higher correlation end regression coefficients (Table I). The 2nd end 3rd follicles are under-estimatedbecause they are not found when their diameter is less than 8 mm. 3. - Preovulatory follicular growth estimated by ultrasonic echography Follicles were measured in 6 mares during the follicular phase of the oestrous cycle. Fig. 1 shows the mean curve of development of the two largest follicles. Three interestingpoints may be noted in this study: l- The largest follicle stopped its growth one day before ovulation, 2- The second largest follicle was not growing during the whole period. 3- The follicle due to ovulate was already the largest about 6 days before ovulation. These findings suggested the possibility of estimating the interval to ovulation from the measurement of the size of the largest follicle. This hypothesis was tested in a group of 15 mares submitted to a progestagen treatment followed by prostaglandin injections to synchronize onset of follicular phase (3). Measurements of follicles were made on the day of the prostaglandin injection and 4 days later. All measurements were plotted against interval to ovulation (Fig. 2). The correlation and regression of follicular measurement X on interval to ovulation Y were highly significant (r= -0.70) but insufficientto give a precise estimate of the time of ovulation. However, we were able to predict if an animal would ovulate early or late within the group studied. 4. - Visualization of the conceptus in utero by echography A vertical axial section of the organs situated under the rectum will show successively the vagina, the cervix and the uterine body. The observer needs to be experienced

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to distinguish these structures (Photo 3). The bladder and the pubis are easy to recognize ventrally. When the anterior end of the uterine body has been reached, moving the probe laterally will give a cross section of the uterine horn, whereas the uterine body will disappear (Photo 4). In pregnant mares, a lo-15 mm spherical vesicle full of liquid is visualized in one horn (near the junction with the body) as early as day 14 post-ovulation (Photo 5). The appearance of the conceptus was studied in a group of 8 regnant mares for a total of 39 observationsbetween day 1E and day 63 of pregnancy. The growth of the vertical diameter and the main changes in the shape of the conceptus during this period are summarized in Fig. 3: the diameter increases rapidly from day 14 to day 20, and stops growing at around 20-25 mm for 10 davs. During the period from day 20 to 25 the s-herical aspeci of the ckceptus is not so clear (Photo 6P . During the period between day 25 and 30 (Photo 7) a spot and/or a membrane were found inside the liquid and were interpretedas the first picture of the embryo or development of the allantoic sac (4). From day 30 the embr o is clearly distinguishableinside the vesicle (Photo 83 , and the vesicle increases in size again. The vertical diameter increases from 25-30 mm on day 30 to 90100 mm on day 60. Around day 55 the embryo is not found floating in the vesicle, but appears at the bottom and shows strong movements at irregular intervals. 5. - Validation of early pregnancy testing by ultrasonic echography 84 early pregnancy tests were performed between day 14 and day 21 in different stud farms without knowledge of whether return to oestrus had occurred. Negative diagnoses were confirmed by return to oestrus or low progesterone before day 45 or a negative PNISGtest at 45 days. Positive diagnoses were confirmed by PMSG tests after day 45 and plasma total conjugated oestrogen assay after day 85 (2). In 44 cases the mares were diagnosed not pregnant by early echography. Of these, only three were in fact pregnant, All three false negative diagnoses were performed during our learning period, One occurred on day 16 and two on day 21 of pregnancy. The error can not be attributed to testing too early, since on day 21 the conceptus begins to loose its spherical appearance and becomes more difficult to see than between days 15-20. All other negative diagnoses were confirmed either by return to oestrus or a period of low progesterone or absence of PMSG. In 40 cases the mares were diagnosed pregnant, An oestrogen test performed later than day 90 confirmed the positive diagnosis in 36 (90%) of them. Of the four false

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positive, two were under serial blood sampling and both showed a maintained corpus luteum, suggesting that pregnancy had occurred followed by embryonic mortality. The third showed a very distinct spherical vesicle on day 15, but it had disappeared on day 19. Progesterone was high until day 18, suggesting a slightly prolonged luteal phase. The fourth case was found in a mare on day 20, but no progesterone data were available. The mare was found not pregnant in a PMSG test on day 90 and in an oestrogen assay performed later. The overall accuracy of our 84 diagnoses was 92%. In a more recent study, 70 inseminatedmares were tested independentlyon day 15, day 3021, and day 45+1, Forty mares were diagnosed pregnant on day 15 and thirTy not pregnant. Day 30 and 45 observations confirmed the day 15 observations in all animals except one which was positive on day 15 and negative on day 30 and 45. Again in this mare the C.L. persisted indicating probable fecundation and embryonic mortality. DISCUSSION In the human, visualization of the uterine content is now classic (5), but visualization of ovarian follicles is more recent (6). Ultrasonic echography was not possible for equine gynecologic examination until a real time echoscope with a small mobile probe became available, allowing per-rectum observation. During preliminary experiments visualization through the flank was tried, but was unsuccessful. Examination of follicles of the mare is easy because the size of the follicles is much bigger than the resolution of the apparatus (8-10 mm). In cattle, where follicles are smaller, only PMSG stimulated ovaries can be seen (7). Our examinations of ovarian follicles were of equal or better quality than rectal palpation, whereas we had much more experience in rectal palpation than in echography, The comparison would probably be more favorable to echography after longer experience with the technique. In addition, the technology of echoscopes is rapidly improving and one can expect better resolution and more accurate estimations in the future. Underestimationof 2nd and 3rd follicles should be reduced. Another contribution of echography which was not shown by this study is differentiationbetween the young corpus luteum (about 36h of age) and a very soft follicle. In some instances palpation will not give convincing evidence, especially if this structure is not the largest in the ovary

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and if palpation was not performed at 24h intervals. Echography will give en unquestionableanswer, the young CL ~.. giving a high echo and the follicle no echo at all. Our studies of follicular growth have confirmed some findings obtained by rectal palpation of the ovaries (8). Echography is already used in our routine palpation experiments when any doubt exists about ovulation of a small follicle or when using small ponies with a rectum of small diameter. However, for practical application, the greatest contribution is the visualization of the early conceptus. Its spherical aspect seems a peculiarity of the equine and human species and allows an early diagnosis (day 14 postovulation in the mare and day 20 in the woman). In the cow where the conceptus is elongated, it is necessary to wait until day 30 to see a liquid phase inside the uterus (7). The size of the vesicle found is in agreement with descriptions of the conceptus in the literature (4,9). In all cases, as early as day 15 the conceptus was found in the same position, in one horn near the body junction, Myometrial contractions are probably responsible for this standardized position. This constant position of the conceptus makes it easy to find and after the insertion of the probe, usually no more than one minute is necessary to perform the diagnosis. The negative diagnoses from tests performed as early as day 15 post-ovulation are sufficientlyaccurate to allow treatment of animals to induce return to oestrus. When compared to progesterone assay, echography has the foil.owingadvantages: diagnosis can be made earlier (day 14 vs. 18), no specific time is required (any time after day lb), there is no error due to a persistent CL, and there is no delay between sampling and answer. When compared with rectal palpation of uterine tone, the advantages are: earlier diagnosis, no error due to persistent CL (lo), probably more accurate although this was not critically tested, and the technique can be learned rapidly (one or two days of learning are sufficient to perform pregnancy diagnoses), As for PMSG tests, they may be more accurate but they are performed much later and consequently are not affected by the embryonic mortality occurring between day 15 and 45.

We hope to be able to visualize twin pregnancies as it has been done in the human (11). However, the first studies in mares that presented twin ovulations did not show two separate vesicles in the uterus, In some instances we suspected that two vesicles were adjacent, but confirmation has not been obtained,

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Three cases of abortion of twins have occurred in the mares of the first study in which no twins had been detected. Further studies are necessary to elucidate why one conceptus is so easy to detect and two are not. The incidence of early embryonic mortality, which explains easily the 11% false positive diagnoses found, should be reexamined in the future with the help of this technique. CONCLUSION Echography is a new approach for the examination of the genital tract of the mare, It gives an improvement of estimation of follicular population when one is interested in other than the largest follicle. It allows an early pregnancy diagnosis with a good accuracy. The application of this technique in veterinary medicine may be as important as in human medicine,

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REFERENCES

1. Palmer, E., Thimonier, J. and Lemon, M. Early pregnancy diagnosis in the mare by estimation of the level of progesterone in the peripheral blood. Livestock Production Science 1 : 197-206 (1974). 2. Terqui, M. and Palmer, E. Oestrogen pattern during early pregnancy in the mare. J. Reprod. Fert. 11 (Suppl.) : 441-446 (1979). 3. Palmer, E. Different techniques for synchronizationof ovulation in the mare. Proc. 8th Intern. Congress of Anim. Reprod. and A.I., Krakow. Pp. 495-498 (1976). Van Niek@rk, C.H. and Allen, W.R. Early embryonic development in the horse. J.Reprod. Fert. 12 (Suppl.) : 495-498 (1975). 5. Levi, S. and Erbsman, F. La croissance du sac embryonnaire humain Btudiee par ultrasons. Rev. Franc. Gynec. 69 : l-12 (1974). 6. Hackeloer, B.J. The ultrasonic demonstration of follicular development during the normal menstrual cycle and after hormone stimulation. Proc. Internat. Sympos. Recent Advances in Ultrasound Diagnosis, Dubrovnik. Pp. 122-128 (1977). 7. Chupin, D. Diagnostic precoce de gestation et de nombre de foetus chez la vache par &hotom.ographie.Ann. Zootechn. (submittedfor publication). a. Nishdlcawa,Y. Studies on reproduction in horses. Japan Racing Association, Tokyo. (1959). 9. Marrable, A.M. and Flood, P.F. Embryological studies on the Dartmoor pony during the first third of gestation. J. Reprod. Fert. 23 (Suppl.) : 499-502 (1975). 10. Kooistra, L.H. and Ginther, O.J. Termination of pseudopregnancy by administration of prostaglandin F2Mand termination of pregnancy by administrationof prostaglandinF2@ or colchicine or by removal of the embryo in mares. Am.J.Vet.Res. 37 : 35-39 (1976). 11. Levy S. Ultrasonic assesment of the high rate of human multiple pregnancy in the first trimester. J. Clin. Ultrasound. 4 : 3-5 (1978).

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EVOLUTION

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FIGURE

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Photo NoI : echographic view of an ovary with 5 follicles of 28, 28, 15, 10, 8 mm respectively. Squares give the scale (I cm)

Photo No2 : echographicview of an ovary with two young corpora lutea I and 3 days old (white areas) surrounded by several 8-15 mm follicles (black areas)

Photo No3 : longitudinal section of uterine body. Elongated dark area is uterine body. White area at the bottom is the pubis bone.

Photo No4 : echographic view of cross-section of uterine horn (round dark but not black area) near junction with uterine body.

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Photo No6 : Day 24 conceptus has lost its spherical aspect but is characterised by liquid-phase (black) sharply contrasting with uterine tissue (prey). It is usually found at the same place as previous photographs.

Photo No7 : Day 25 conceptus with supposed embryo (white area at the bottom) and supposed allantoic membrane (white dotted line in the upper right part).

Photo No8 : Day 33 conceptus with distinct embryo (white spot) floating inside the liquid (black area).

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