The ability to determine ovarian activity during normal estrous periods or in response to synchronization or ovulatory induction protocols is critical for the development of efficient and repeatable artificial insemination protocols. Ultrasonographic ovarian imaging has been used extensively and effectively with domestic species for this purpose. In this study, we utilized transabdominal ultrasonography to evaluate the ovarian response to a previously reported ovulatory induction protocol which utilized the exogenous gonadotropins equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) (Sawyer-Steffan et al. 1983; Schroeder Keller, 1990). Our objectives of this study were to: 1) determine our ability to serially locate and evaluate dolphin ovaries with transabdominal ultrasound; 2) to compare ovarian image quality (i.e., resointion) obtained from 3 different ultrasound transducers (3.5 MHz Linear, 5 Ml-Iz convex linear, 5 MHz fincar); 3) to evaluate the ovarian response to an ovulation induction protocol utilizing eCG and hCG; 4) determine and compare the echogenic pattern of the uterus during the follicular (estrogenic period) and luteal (progestogenic period) phases of the Cycle.

Two sexually mature parous female bottle-nosed dolphins, Tursiops truncatus, located at Sea World of California, San Diego was utilized for these procedures. Blood samples were collected and circulating progesterone concentrations were determined 4 and 2 weeks prior to the beginning of the induction protocol. Based on low (<100 pg/nil) serum progesterone concentrations obtained from these samples, the animals were determined to be open and anestrous. The ovulation induction protocol was based on the protocol reported by Schroeder, 1990. However, PG600 was utilized for follicular recruitment and maturation instead of eCG. PG600 (Intervet America Inc., Millsboro, DE) has 400 TU eCG and 200 IU hCG/5 nil of activity and is dosed based on eCG activity. Table I demonstrates the blood sampling and ultrasound periods. Serum progesterone was determined after the examination period. Total immunoreactive estrogens "I be determined. Ultrasonographic follow-up procedures were performed 14 days after circulating progesterone [P] levels had risen above I ng/ml.

Table 1: Treatment, Sampling and Ultrasound Schedule

¹Profasi®, Serono Lab. Inc., Randolph, MA

Ovaries were sonogramed bilaterally from both animals throughout the examination period. The ovaries were located by identifying the caudal pole of the ipsilateral kidney then moving posteriorly (Stone, 1990). Multiple follicles of similar size, approximately I I mm, were initially identified. The maximum follicular size observed by day 9 was 19 nun in dolphin 1. Ovulation was not observed during this initial examination period. However, the large numbers of follicles made it difficult to follow the progression or "disappearance" of individual follicles which may have ovulated. Follicular characteristics did change, from relatively thin walled anechoic spherical structures to irregular shaped structures with hypertrophied walls. Serum progesterone concentrations remained < 200 pg/ml (except for animal I which had 720 pg/ml on day 4) through day 6. By day 8, both animals had elevated serum progesterone concentrations (4560 pg/ml and 1430 pg/ml for animals I & 2 respectively). This increase in serum P corresponded temporally to the observed increase in follicular wall thickness and irregular follicular margins. These changes may have reflected follicular atresia (with or without granulosal or thecal cell luteinization), follicular luteinization, or early post ovulation. Luteal structures were not positively identified. The uterine horns and body were identified and characterized as hypertrophied and edematous.

By day 12, serum P rose to 32,390 and 26,490 for animals I & 2 respectively. Follow up ultrasound examinations on day 22 were performed. Ovaries from both animals had dramatic, multi-follicular development (> 10/ovary) with the largest follicles ranging in size from approximately IS to 3 5 nun. Corpus luteal structures could not be positively identified. In light of the concurrently high circulating progesterone concentrations, the largest follicles could only be characterized as pathologic, luteinized (partial or complete) ovarian cysts. The uterus was echogenically homogenous without signs of edema or hypertrophy. The 17 cm 3.5 MHz linear transducer provided equal or improved image quality when compared to the other transducers, and allowed for simultaneous visualization of the caudal pole of the kidney and the ovary, thus simplifying our ability to locate the ovary.

The results are the first to unequivocally demonstrate that bottle-nosed dolphin ovarian follicular activity can be monitored with ultrasonography. They also demonstrate the potential for misinterpretation of ovarian responses to ovulatory synchronization or induction protocols if methods of protocol evaluation are based only on reproductive steroid profiles. Positive identification of luteal structures was not accomplished. However, serial monitoring of normal ovulatory cycles should yield valuable information concerning corpus luteal formation during conceptive and non-conceptive cycles.

References

1.  Sawyer-Steffan JE, Kirby VL Gilmartin WC. 1983. Progesterone and estrogens in the pregnant and non-pregnant dolphin, Tursiops truncatus, and the effects of induced ovulation. Biol. Reprod. 28:897-901.

2.  Schroeder, J.P. Reproductive Aspects of Marine Mammals. In: CRC handbook of marine mammal medicine: health, disease, and rehabilitation, edited by Dierauf, L.A. Boca Raton: CRC Press, 1990, p. 353-369.

3.  Schroeder, J.P, and Keller, K.V. Artificial insemination of bottlenose dolphins. In: The bottlenose dolphin, edited by Leatherwood, S. and Reeves, R.R San Diego: Academic Press, 1990, p. 447-460.

4.  Stone, L.R. Diagnostic ultrasound in marine mammals. In: CRC handbook of marine mammal medicine: health, disease, and rehabilitation, edited by Dierauf, L.A. Boca Raton: CRC Press, 1990, p. 235-264.