Characterization of Follicular Recruitment and Growth Using Endocrine and Ultrasound Evaluation in the Bottlenose Dolphin (Tursiops truncatus)
Ultrasonographic exams of follicular development and hormone assessments of bottlenose dolphins that did and did not ovulate following estrus synchronization (termed ovulators and non-ovulators, respectively), and of conceptive and non-conceptive ovulators, were performed to characterize and compare the reproductive physiology of these states using a mixed model repeated measures regression analysis with animal ID as the random variable. Daily follicle measurements were grouped by follicle size in mm (very small [VSM], small [SM], medium [MD] and large [LG]). Upon its first identification at 9.7 days (± 0.74 d) prior to ovulation (DPO), the dominant follicle (F1) grew 1.3 ± 0.2 mm/day, with a mean maximum size of 18.4 ± 0.6 mm at 12–24 hours prior to ovulation. No differences (p > 0.05) were detected between the total follicle numbers on the side contralateral or ipsilateral to the ovulation. There was a significant effect of DPO on numbers of VSM, MD and LG (p < 0.001), but not SM. Initial follicular wave peaks comprised of different follicle sizes were detected prior to identification of F1 at 17 (VSM), 15 (SM) and 14 (MD) DPO. Establishment of follicular dominance had a negative effect (p = 0.01) on total follicle number. Age was correlated (p < 0.05) with peak number of SM, and MD, but not with VSM number or pre-ovulatory follicles. F1 growth rate did not affect conception rate (p = 0.33); however, animals with a live calf had significantly (p < 0.05) increased F1 growth rate and a shorter time from peak VSM to ovulation. The non-ovulator group presented significantly lower concentrations of urinary follicle stimulating hormone (FSH) and estrone conjugates (E1-C), but similar estriol (E3), progesterone and luteinizing hormone (LH) concentrations compared to ovulators. Urinary concentrations of E1-C, E3, and numbers of MD and LG increased (p < 0.05), and FSH concentration decreased (p < 0.05) as ovulation approached. Urinary LH significantly increased with increasing numbers of LG (while SM numbers decreased). No differences were detected in serum E3, Activin A (A) or anti-Müllerian hormone (AMH) concentrations between ovulators and non-ovulators across DPO, or between or within ovulators and non-ovulators pre- or post-F1 dominance. However, serum FSH concentrations were significantly lower in ovulators than non-ovulators, and serum inhibin A (IA) concentrations significantly decreased after day 12 post-Regumate in ovulators. Serum E3 and A concentrations significantly increased as ovulation approached and E3 concentration was positively related (p < 0.05) to MD and LG number. Serum AMH concentrations decreased (p < 0.05) with increasing numbers of MD follicles. Follicle numbers were not influenced (p > 0.05) by serum FSH, E3 or IA, nor were any relationships detected among those hormones. Significant pairwise correlations were detected between serum AMH and A, and for serum IA with AMH, FSH, and E3. The characterization of follicular development and its relationship with hormone assessments complements our understanding of follicular recruitment in mature bottlenose dolphin and provides new information on estrous cycles for use in the species' health assessment, or reproduction and conservation management.
The authors thank the curatorial, training and veterinary staff at Dolphin Research Center, SeaWorld San Diego and Orlando (especially Jesse Pottebaum) for ultrasounds, blood sample collection and processing, and the SeaWorld and Busch Gardens Reproductive Research Center staffs and intern students for hormone assays. The authors are also grateful to Mr. Brad Andrews of SeaWorld Parks and Entertainment (SEA) for continued support of this research.
* Presenting author