Abstract

Non-invasive endocrine monitoring has been used to assess reproductive activity in captive cetaceans, most notably killer whales (Orcinus orca).^1,2 However, past studies relied on radioimmunoassays (RIA) for endocrine analysis. For most marine mammal-holding institutions, the requisite use of radioisotopes, and expensive instrumentation, has precluded routine on-site endocrine monitoring. Alternative non-radiometric enzyme immunoassays (EIA, also known as ELISA, enzyme linked immunosorbent assay) are now available, and these methods are relatively inexpensive and do not require extensive instrumentation. EIA reagents are non-hazardous, easily transportable, disposable, and hormone determinations can be completed within three to four hours. The Conservation and Research Center (CRC) and SeaWorld have established a Mobile Endocrinology Laboratory for conducting on-site endocrine analyses in captive cetaceans. The laboratory monitors urinary estrogen conjugates to optimize the timing of artificial insemination (AI) in cetaceans. Urinary endocrine monitoring, used in conjunction with follicular ultrasound examination, has been key to the successful conceptions by AI in killer whales (n = 3), bottlenose dolphins (n = 2, Tursiops truncatus), and Pacific whitesided dolphins (n = 3, Lagenorhynchus obliquidens).

The Mobile Endocrinology Laboratory employed a single antibody, direct EIA for on-site urinary estrogen monitoring. A luteinizing hormone (LH) EIA, conducted off-site, was used to retrospectively confirm ovulation in all females. Urine samples were collected daily during the study interval, and twice daily when rising estrogen conjugates concentrations suggested the onset of ovarian follicular activity. Female subjects were from four facilities: one killer whale, one bottlenose dolphin, SeaWorld of California, San Diego; one Pacific white-sided dolphin, SeaWorld Texas, San Antonio; one bottlenose dolphin, U.S. Navy Marine Mammal Program, San Diego; and one bottlenose dolphin, Dolphin Quest, Hawaii.

In the killer whale, urinary estrogens (above 1 ng/mg Cr) increased gradually over a ~ten-day interval to pre-ovulatory peak concentrations (6.5 ng/mg Cr), and then declined rapidly to baseline within ~2.5 days (Fig. 1). AI was performed repeatedly before the pre-ovulatory estrogen peak, and the female was inseminated twice following the pre-ovulatory estrogen peak (arrows, Figure 1). Ultrasound confirmed that ovulation (i.e., the rupture of the dominant preovulatory follicle) coincided with peak or falling urinary estrogen concentrations. In the Pacific white-sided dolphin, two ovarian cycles were monitored. The first cycle was after ovulation induction with altrenogest (Regu-Mate®, Intervet Inc., Millsboro, DE 19966 USA), and the second was the subsequent spontaneous ovarian cycle (Figure 2). During the induced cycle, urinary estrogen excretion increased over a ~six-day interval to peak pre-ovulatory concentrations, and then declined to nadir two days later. A similar profile was observed in the subsequent ovarian cycle: urinary estrogens increased over a ~eight-day interval to a preovulatory peak (1.8 ng/mg Cr), followed by an abrupt decline to baseline. Peak urinary LH excretion was observed approximately 17 hours after the estrogen peak, and this coincided with rupture of the dominant ovarian follicle, which was confirmed directly by ultrasound examination. These data suggest that AI timing was optimal during a ~24-hour interval beginning with the pre-ovulatory urinary estrogen peak. In one bottlenose dolphin (SeaWorld, San Diego), urinary estrogens increased ~12 days before the pre-ovulatory estrogen peak (3 ng/mg Cr) and returned to nadir levels within 2.5 days. Similar trends were observed in another bottlenose dolphin (Dolphin Quest, Hawaii), in which two ovarian cycles were monitored (Figure 3). During the first ovarian cycle, peak urinary estrogen and LH excretion occurred simultaneously, whereas in the second cycle, peak urinary LH occurred ~12 hours after the urinary estrogen peak. Although more data is needed, these results suggest that, like the Pacific white-sided dolphin, the bottlenose dolphin's AI timing is probably optimal during the 12-24 hour-interval that begins with the pre-ovulatory urinary estrogen peak. Although, urinary estrogen excretion in the first of two ovarian cycles monitored in another bottlenose dolphin (US Navy, San Diego) were typical of trends observed previously, post-ovulatory decline in urinary estrogen to baseline was prolonged (~ nine days). This same female exhibited an irregular estrogen excretion profile during the subsequent cycle, which suggested anovulation.

These data suggest that on-site endocrine evaluations provide a rapid, cost-effective, and accurate means for assessing reproductive-endocrine activity in cetaceans. The validity of estrogen excretion measures was confirmed by the close correspondence between the preovulatory LH surge, as well as direct observations of ovarian follicles by ultrasound. Although more data are needed, these results demonstrate the tremendous potential of noninvasive endocrine monitoring for improving our understanding of the reproductive biology of cetaceans. Finally, these results demonstrate that a Mobile Endocrinology Laboratory, combined with serial ultrasonography, provides a powerful combination for ensuring the success of AI in cetaceans.

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	Figure 1. Urinary estrogen conjugates in the killer whale
	Figure 2. Urinary estrogen conjugates and LH in the Pacific white-sided dolphin
	Figure 3. Urinary estrogen conjugates and LH in the bottlenose dolphin
	Figure 4. Urinary estrogen conjugates in the bottlenose dolphin

Acknowledgements

We thank trainer and laboratory staff at SeaWorld California, SeaWorld Texas, US Navy Marine Mammal Program, and Dolphin Quest, Hawaii for assistance with sample collection.

References

1.  Robeck TR, Schneyer AL, McBain JF, Dalton LM, Walsh MT, Czekala NM, Kraemer DC. 1993. Analysis of urinary immunoreactive steroid metabolites and gonadotropins for characterization of the estrous cycle, breeding period, and seasonal estrous activity of captive killer whales (Orcinus orca). Zoo Biology 12, 2:173-188.

2.  Walker LA, Cornell L, Dahl KD, Czekala NM, Dargen CM, Joseph BE, Hsueh AJW, Lasley BL. 1988. Urinary concentrations of ovarian steroid hormone metabolites and bioactive follicle-stimulating hormone in killer whales (Orcinus orca) during ovarian cycles and pregnancy. Biology of Reproduction 39:1013-1020.