Characterization of Reproductive Cycles and Development of an Ovulation Induction Method in the Beluga (Delphinapterus leucus)
Much of the knowledge and information regarding beluga biology and reproduction has been obtained from in situ studies or post-mortem sampling of harvested animals (Brodie, 1971; Braham, 1984; Burns and Seaman, 1986; Heide-Jørgensen and Teilmann 1994). Despite these efforts, in situ research is often limited to infrequent sampling, leaving many physiological questions unanswered. A thorough understanding of reproductive function and elucidation of specific mechanisms typically requires daily, even hourly sample collection. In an effort to answer these questions and thus improve the success of captive management procedures, a cooperative captive beluga breeding program amongst zoological institutions and aquariums was initiated in 1988 (Robeck et al., 2005). The program resulted in the successful birth of over 20 calves, and regular sample collections and behavioral observations made during this period were used to answer many questions concerning beluga reproduction (Robeck et al., 2005). However, despite these successes, the total captive population of beluga remains small with animals distributed across many facilities. Assisted reproductive technologies (ART), such as artificial insemination (AI), represent a means of maximizing genetic management of these small populations. However, the development of AI requires another level of understanding of basic reproductive biology prior to successful implementation.
The objectives of the current research were to define the basic reproductive endocrinology of the female beluga estrous cycle and develop an ovulation induction method. Daily urine samples were collected from 2002 to 2005 from four female beluga housed at SeaWorld of Texas (SWT, San Antonio, TX). A breeding male was only present in the environment during the last year of the study (2005). Samples were analyzed for urinary concentrations of estrogen conjugates (uEC), luteinizing hormone (uLH), and progestagens (uPg). Transabdominal ultrasound was performed using a 3.5 mHz curvilinear probe (GE LogiqTM Book, GE Medical Systems, Milwaukee, WI). Ultrasound data were obtained weekly to three times weekly, depending on the stage of the estrous cycle. Data are presented as mean ± sd unless otherwise indicated. From 2002 to 2004, ten estrous cycles were evaluated. Urinary estrogen conjugates increased above baseline concentrations for a period of 13 to 29 days (21 ± 6 days). Estrogen peaks (64 ± 54 ng/mg Creatinine [Cr]; range: 46-201 ng/mg Cr) were associated with presence of a dominant follicle reaching a maximum circumference of 8.6 ± 2.4 cm. In the absence of a breeding male, follicles regressed and urinary EC returned to baseline concentrations after a period of 8 to 59 days (24 ± 15 days). For nine of the ten cycles analyzed, no coinciding ovulatory uLH surges were detected with uEC peaks, and no increases in uPg were observed thereafter. In one beluga, uEC rose above baseline for 19 days, peaking at 71 ng/mg Cr. This period was associated with an ovulatory LH surge of 16 ng/mg Cr and was followed by increase in uPg (for 23 days, peak 7 ng/mg Cr) after ovulation. The inter-estrus interval for this female was approximately 35 days. In 2005, a breeding male was introduced to the SWT female beluga prior to the commencement of the breeding season. Seven estrous cycles were observed during 2005. Urinary EC increased above nadir levels for 13 to 26 days (20 ± 5 days) and returned to baseline within three to 20 days (8 ± 7 days). Six of the cycles were designated as ovulatory due to detection of a corresponding uLH surge (20 ± 7 ng/mg Cr) and subsequent increase in uPg. Two of the seven ovulatory estrous cycles resulted in pregnancy.
Based on these results it was hypothesized that beluga were facultative-induced ovulators and that ovulation induction techniques would be required for successful AI. Accordingly, the effectiveness of an exogenous gonadotropin releasing hormone (GnRH; Cystorelin®, Merial, Duluth, GA) to induce ovulation was evaluated. A total of seven trials with three beluga located at SWT and SWC (SeaWorld California, San Diego, CA) were conducted. All animals exhibited behavioral estrus and had growing follicles prior to GnRH administration. Animal one received a single dose of 100 µg GnRH i.v. without an effect. Follicular circumference just prior to GnRH administration was 10.5 cm. In the second trial, two 250 µg doses of GnRH were administered four hours apart to a beluga with a follicular circumference of 8.0 cm. The animal ovulated between 24 and 48 hours post-injection. The remaining five trials (in two animals) utilized three 250 µg doses of GnRH, given either three (n = 3) or two (n = 2) hours apart. Pre-administration follicular circumference was 7.89 ± 1.97 cm (range: 5.74 to 10.53 cm). All beluga ovulated within 33.7 ± 2.3 h (range: 31.5 to 36 h) after the first GnRH injection. These data suggest ovulation induction can be successfully achieved via administration of an exogenous GnRH in the beluga. The information acquired from the present research will facilitate the development of ART in the beluga, and ultimately enhance captive breeding efforts for this species.
The authors would like to thank Mr. Brad Andrews and Busch Entertainment Corporation for support. We thank Dr. Steven Monfort of the Smithsonian's National Zoological Park for his support. Additionally, we thank the veterinary, animal training and laboratory staff at SeaWorld Texas, and SeaWorld California for their assistance with sample collection. In particular we thank Dr. Todd Schmitt (SWC), Doug Acton (SWT), Chris White (SWT), and Brent Posey (SWT) for their dedication to these efforts.
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