R.A. Pierson

Ultrasonography of the bovine ovary

A linear-array ultrasound scanner with a 5-MHz transducer was evaluated for the study of follicular and luteal status in heifers. The ovaries of five heifers were monitored daily until all heifers were examined for a period from three days before an ovulation to three days after the next ovulation. There was a significant difference among days for diameter of the largest follicle and second largest follicle and for the number of follicles 4-6 mm and >10 mm. Differences seemed to be caused by the presence of several 4- to 6-mm follicles in early diestrus, growth to an ostensibly ovulatory size and subsequent regression of a follicle during mid-cycle, and selective accelerated growth of the ovulatory follicle four days before ovulation. The corpus luteum became visible approximately three days after ovulation and was identifiable throughout the rest of the interovulatory interval. In two of the five heifers, the corresponding corpus albicans was identified for three days after the second ovulation. Two heifers were induced to superovulate and follicular growth was monitored. The results indicated that the follicles which ovulated originated from the population present when the superovulation treatment was initiated. The ultrasound instrument was judged effective for monitoring and evaluating ovarian follicles and corpora lutea in normal and superovulated heifers.

Ultrasonic morphology of corpora lutea and central luteal cavities during the estrous cycle and early pregnancy in heifers.

Ultrasonography was used once daily to quantify corpora lutea, central luteal cavities, and luteinized tissue during interovulatory intervals (n=66) and during Days 0 to 60 of pregnancy (n=14) in nulliparous Holstein heifers (ovulation=Day 0). The corpus luteum of the estrous cycle was detectable by ultrasonography in most heifers from the day of ovulation (mean, Day 0.5) and extending into the regressive phase beyond the next ovulation (mean, Day 1.4+/-0.2 after the next ovulation). During pregnancy, the corpus luteum was detected until Day 60 (end of study). Maximal central luteal cavity area detected on Days 0 to 20 was used retrospectively to group luteal glands into four cavity categories: no, small, medium, and large. These categories corresponded to approximate cavity diameters of <2 mm, 2 to 5 mm, 6 to 10 mm, and >10 mm, respectively. The incidence of each cavity category was similar between interovulatory intervals and pregnancies (combined incidence, 17/80, 8/80, 33/80, and 22/80 for no, small, medium, and large cavities, respectively; total with cavities, 63/80, 79%). Mean day of first detection of a central cavity was earliest for large cavities during interovulatory intervals (means, Days 4.7, 4.4, and 3.0 for small, medium, and large cavities, respectively; P<0.04) and during pregnancies (means, Days 5.5, 4.2, and 3.3, respectively; NS). However, the day that the cavities reached maximum size (range of means, Days 5.5 to 7.0) did not differ among categories. Mean day of last detection of the central cavity was significantly different among cavity categories during interovulatory intervals (means, Days 9.3, 11.1, and 17.4 for small, medium, and large cavities, respectively) and pregnancies (means, Days 7.0, 8.8, and 20.2, respectively). Time of loss of central cavities was similar between nonbred and pregnant heifers, and there was no significant difference among cavity categories in the length of the interovulatory interval (mean, 20.1 d). Luteal tissue area was not significantly different among cavity categories during interovulatory intervals. There were no indications that cavities were functionally important. Luteal tissue area increased linearly in pregnant heifers on Days 21 to 60 (mean slope, 2.6 mm2/day).

Ultrasonography for detection of pregnancy and study of embryonic development in heifers

Real-time B-mode ultrasonography was used to evaluate uterine changes associated with the estrous cycle in 22 ovulatory periods in 12 nulliparous heifers. Irregular, nonechogenic (black) areas were seen on the images of uterine horns during the periovulatory period. These nonechogenic areas were presumably due to intraluminal fluids since they coincided with the discharge of clear, viscous mucus preceding ovulation and blood-tinged mucus after ovulation. Eight heifers were bred until five pregnant heifers were obtained for study of the ultrasonic morphology of the conceptus. Ultrasound examinations were done daily to day 50 of pregnancy. Discrete, nonechogenic areas were first visible within the uterus between days 12 and 14, when they were approximately 2 mm in diameter. These discrete nonechogenic structures were identified as the embryonic vesicle, since they were observed only in heifers later confirmed to be pregnant and were always in the uterine horn ipsilateral to the corpus luteum. The presence of an embryo within the embryonic vesicle was confirmed by observing an echogenic (white) area with rhythmic pulsations (heartbeat). The embryonic vesicle gradually increased in length from the day of first observation until day 26 when it extended past the curvature of the horn and began to encroach into the contralateral horn. In all heifers, by day 32 the vesicle extended to the tip of the contralateral horn. The embryo was first visible between days 26 and 29 when the mean length was 10 mm. The embryo increased in length an average of 1.1 mm per day. A heartbeat was detectable in the embryo on the first day observed. In one superovulated heifer, five vesicles were visible in the uterine horns by day 14 and by day 33 seven embryos were observed; two of the seven embryos apparently resorbed by day 43.

Ultrasonic anatomy and pathology of the equine uterus

The morphological and pathological status of the uterus in mares was evaluated using a linear-array ultrasound scanner, and the ultrasonic properties of the uterus were characterized. The uterus was examined each day in 16 mares, beginning at mid-diestrus. The uterus was recorded as having an ultrasonic morphology characteristic of diestrus (endometrial folds not distinguishable), estrus (prominent endometrial folds) or an intermediate stage (folds only moderately distinguishable). The number of mares with an intermediate or estrous image increased gradually between day -7 (2 14 mares; ovulation = day 0) and days -3 (11 16 ) and -2 (10 16 ) and then declined between days -2 and +1 (0 12 ). In another study, a large nonechogenic area (ejaculate) was visible in the uterine lumen immediately after mating in all of six mares. During the course of pregnancy diagnoses and hand-breeding, several pathological conditions of the uterus were first observed by ultrasound examination and confirmed by digital exploration of the uterus or at slaughter. Cysts contained nonechogenic material (fluid) and were usually compartmentalized. The purulent material associated with pyometria was relatively nonechogenic but contained echogenic spots. Mummified fetuses and remnants of fetal bones showed echogenic properties consistent with high tissue density. The results demonstrated that ultrasound technology provides a noninvasive form of visual access to the uterus to evaluate normal, morphological changes and to detect and study certain pathological processes.

Reliability of diagnostic ultrasonography for identification and measurement of follicles and detecting the corpus luteum in heifers

Abstract The accuracy of diagnostic ultrasonography for assessment of ovarian structures was examined by comparing results of in vivo ultrasonography and slices of the excised ovaries. Follicular numbers and diameters, location (right versus left ovary) of the corpus leteum, and presence of fluid-filled cavities within the corpus luteum were evaluated in 23 Holstein heifers on Days 12 or 14 postovulation. The following endpoints were used for each ovary (n = 46): number of follicles 2 to 3 mm, ≥4 mm, ≥7 mm, and ≥11 mm and diameter of largest follicle. Heifers were slaughtered within 4 hours of ultrasound examination. Ovaries were collected and placed in 10% formalin for 12 hours to prevent collapse of the follicles. Slices 2 mm thick were made to expose the follicles. Slicing determinations were made without knowledge of ultrasound results. Comparisons were done by regression and correlation analyses and paired t-tests. The 95% confidence intervals revealed a tendency to slightly overestimate the number of 2 to 3 mm follicles (ultrasonography, 16.4 ± 0.7 SEM; slicing, 15.5 ± 0.8). Zero was centered within the confidence intervals for the number of follicles ≥4 mm, ≥7 mm, and ≥11 mm and diameter of largest follicle. Slopes of the regressions for number of follicles determined by ultrasonography versus slicing ranged from 0.83 for number of follicles ≥4 mm to 1.03 for number of follicles ≥2 mm. Regression R 2 values were from 64.5% to 84.9% for various categories and 94.5% for diameter of largest follicle. Correlation coefficients between ultrasonography and slicing results were highly significant (number of follicles 2 to 3 mm, 0.90; ≥4 mm, 0.80; ≥7 mm, 0.89; ≥11 mm, 0.85; ≥2 mm, 0.92 and diameter of largest follicle, 0.97). There was 100% agreement between ultrasound and slicing results for identification of the corpus luteum bearing ovary and presence of cavities within the luteal gland. Diagnostic ultrasonography was determined to be a reliable method of identifying and measuring follicles and detecting mature corpora lutea and luteal cavities in heifers.

Ultrasonic evaluation of the preovulatory follicle in the mare.

Ultrasonically visible characteristics of preovulatory follicles in mares which single ovulated were studied daily for 79 preovulatory periods in 40 mares. The preovulatory follicle became the largest follicle in the ovary from which ovulation later occurred six or more days before ovulation in 65 of 79 (82%) preovulatory periods; the mean was day -7 (range, day -14 to day -4). The increase in mean diameter of the preovulatory follicle was linear (R(2)=99.5%) over day -7 (29.4 +/- 0.8 mm) to day -1 (45.2 +/- 0.5 mm; growth rate, 2.7 mm/day). Follicles which double-ovulated were smaller (P<0.05) on day -1 (36 +/- 1.6 mm; n=12 follicles). Preovulatory follicles exhibited a pronounced change in shape from a spherical to a conical or pear-shaped structure in 84% of the preovulatory periods. Remaining follicles retained a spherical shape. Scores representing thickness of the follicular wall increased (P<0.05) as the interval to ovulation decreased. There was no significant difference among days in mean gray-scale value of the follicular wall or in echogenicity of the follicular fluid. Although diameter and shape of the follicle and thickness of the follicular wall changed during the preovulatory period, no reliable ultrasonically visible predictor of impending ovulation was found.

Ultrasonic anatomy of equine ovaries.

A linear-array ultrasound scanner with a 5-MHz transducer was evaluated for studying follicular and luteal status in mares, and the ultrasonic properties of equine ovaries were characterized. Follicular diameters were estimated in vivo and after removing and slicing six ovaries. Correlation coefficients between the two kinds of determinations were 0.91 for number of follicles >/=2 mm in diameter and 0.95 for diameter of largest follicle. The ovaries of five mares were examined daily until all mares had been examined from three days before an ovulation to three days after the next ovulation. There was a significant difference among days for diameter of largest follicle and second largest follicle and for number of follicles 2-5 mm, 16-20 mm, and >20 mm. Differences seemed to be caused by the presence of many 2- to 5-mm follicles during early diestrus, initiation of growth of large follicles at mid-cycle, selective accelerated growth of an ovulatory follicle beginning five days before ovulation, and regression of large nonovulatory follicles a few days before ovulation. In one of the five mares, the corpus luteum was identified throughout the interovulatory interval, and the corresponding corpus albicans was identified for three days after the second ovulation. In the other four mares, the corpus luteum was last identified an average of 16 days after ovulation or five days before the next ovulation. In a blind study, the location of the corpus luteum (left or right ovary) as determined by ultrasonography agreed with a previous determination of side of ovulation by palpation in 88% of 40 mares on days 0-14. In the remaining 12% and in all of 12 estrous mares, the location was recorded as uncertain. The ultrasound instrument was judged effective for monitoring and evaluating follicles and corpora lutea.

Ultrasonic evaluation of the corpus luteum of the mare

Two distinct luteal morphologies were observed in the ovaries of mares studied by daily ultrasound examinations. Luteal glands that formed after 48.5% of 95 ovulations were uniformly echogenic over 90 to 100 percent of the area of the image of the gland throughout the period of detectability. The remaining luteal structures (51.5%) exhibited a centrally located nonechogenic area. The nonechogenic area was first detected on day 0 (28%), day 1 (62%), day 2 (6%) or day 3 (4%) postovulation. Glands classified as centrally nonechogenic were echogenic over 80 to 100 percent of the area of the image of the gland on day 0; mean percentages of echogenic tissue decreased to 45 percent by day three then gradually increased to 95 percent before the glands became unidentifiable. The echogenic portion of the luteal glands of both morphologies had a bright echogenicity (gray-scale zone 4.5 to 5) on day 0. The echogenicity decreased (zones 3 to 3.5) by day 8 and was maintained at approximately that level until day 12. Mean gray-scale values tended to increase (zone 4 to 4.5) prior to the time the luteal glands became ultrasonically unidentifiable. These changes in grayscale values may have reflected changes in luteal hemodynamics. The nonechogenic area of centrally nonechogenic glands was attributed to clotted blood (corpus hemorrhagicum). The formation of a corpus hemorrhagicum was apparently not functionally important because it was present in only one half of the luteal glands. In addition, the mean length of time that the luteal gland was identifiable (17 days) or the mean length of the interovulatory interval (21 days) was not significantly different between the two luteal morphologies. Therefore, the hypothesis that the formation of a corpus hemorrhagicum is a necessary step in luteogenesis was not supported.

Basic principles and techniques for transrectal ultrasonography in cattle and horses

Abstract Diagnostic ultrasonography is a powerful tool for evaluating the reproductive tracts in horses and cattle. The technology should be considered for use in the embryo transfer industry. Ultrasound imaging technology provides rapid, non-invasive access to the internal reproductive organs. Dynamic structures may be visualized in the living animal — structures that were previously detectable only in the static state at necropsy or surgical removal. The potential for assessing reproductive structures in the same animals over time has afforded an unprecedented depth to investigations of the dynamic changes in biological structures (e.g., changes in the ovarian follicular population during the estrous cycle and early pregnancy, the process of ovulation, luteal dynamics, and the interactions between the conceptus and uterus). Ultrasound technology is expensive and requires a thorough working knowledge of anatomy and acoustic principles for maximal utilization. The potential, however, is great for future discoveries in both basic and clinical research. We believe that the best is yet to come as basic research utilizing ultrasonography evolves and ultrasonic imaging continues to be incorporated into clinical and research programs.

Ovarian follicular populations during early pregnancy in heifers

Ovarian follicles >/=2mm were studied in 14 pregnant and 14 nonpregnant Holstein heifers by daily ultrasound examinations. There were significant differences among days, from Day 0 (day of ovulation) to Day 21, in the diameter of the largest follicle and the diameter of the second largest follicle in pregnant and nonpregnant heifers. There was an interaction of day and reproductive status (P < 0.001) for the diameter of the largest follicle. Significant differences among days were also observed in the numbers of follicles 2 to 3 mm, 4 to 6 mm, 7 to 10 mm, 11 to 13 mm, and >13 mm, and the total number of follicles >/=2 mm. There was a significant main effect of reproductive status for the number of follicles 11 to 13 mm. An interaction of day and reproductive status was observed for the number of follicles >13 mm, but not for any of the other diameter categories. The effect of reproductive status for number of follicles 11 to 13 mm and the interactions for the number of follicles >13 mm and the diameter of the largest follicle seemed due to the selective growth and ovulation of the follicle destined to ovulate in nonpregnant heifers. The differences in ovarian follicular populations between pregnant and nonpregnant heifers were attributed solely to the presence of a physiological mechanism for the selection of an ovulatory follicle in nonpregnant heifers. There were no significant differences among days for any follicular endpoint during Days 22 to 60 in the pregnant heifers.