Abstract

Since 2005, artificial insemination (AI) of bottlenose dolphins using sex-selected female (X chromosome-bearing) spermatozoa has resulted in the birth of 10 female calves.^1,2 Wide-spread application of this technology in bottlenose dolphins has been slow due to the significant expense incurred when sorting the minimum insemination dose required to achieve a conception rate of at least 50% (200 x 10⁶ progressively motile sperm).^1-3 Development of a low-dose insemination technique would assist in reducing the expense of the sorting process. Spermatozoa from 4 males were collected, sorted into X-enriched samples (91 ± 2.0% purity) and cryopreserved for use during 4 inseminations. Post-thaw, the inseminate volume was reduced through centrifugation (700 g for 20 min) from 4.3 ± 0.5 ml to 1.0 ± 0.2 ml. Sperm concentration increased from 26.1 ± 6.4 x 10⁶ spermatozoa/ml (post-thaw) to 94.5 ± 10.6 x 10⁶ spermatozoa/ml (post-centrifugation). The post-thaw and post-centrifugation progressive motility and viability were 56.2 ± 4.9% and 57.5 ± 2.7% and 57.9 ± 5.4% and 54.1 ± 5.6%, respectively. The start of the luteinizing hormone (LH) surge was estimated in urine samples using a rapid (20 minutes) semi-quantitative canine LH kit (Witness^® Synbiotics Corp. Kansas City, USA).⁴ Urinary LH and estrogen conjugate (EC) concentrations (indexed to creatinine [Cr]) and the timing of the LH surge were later quantified using an enzyme immunoassay.¹ Peak LH and EC concentrations were 197.9 ± 184.0 ng/mg Cr and 6.9 ± 3.2 ng/mg Cr, respectively. AIs were performed 31.3 ± 4.6 hr post-start of the LH surge with a maximum pre-ovulatory follicle diameter of 1.7 ± 0.1 cm. Females were inseminated with 55.7 ± 10.1 x 10⁶ progressively motile sexed spermatozoa. Fifty percent of the X-enriched sample was deposited at the proximal most accessible region (presumptive utero-tubal junction) in each uterine horn, 58.6 ± 10.1 cm beyond the genital opening using a flexible endoscope. The external cervical Os and internal uterine bifurcation were located 22.5 ± 3.5 cm, 33.8 ± 5.9 cm beyond the genital opening, respectively. All but one of the insemination were performed a maximum of 12 hrs prior to ovulation. Two of the 4 inseminations (50%) were conceptive as confirmed by visualization of a fetus using ultrasound. These findings provide preliminary validation of modifications made to the established AI methodology for the bottlenose dolphin. Such modifications have been shown to result in a 4 fold reduction in the insemination dose whilst still maintaining an acceptable conception rate. If consistent results are obtained in future AI attempts, this development will have a significant impact on the efficient use of both sexed and non-sexed cryopreserved bottlenose dolphin.

Acknowledgements

We thank the animal care laboratory, animal training and animal care staff at SeaWorld San Antonio (SWSA), and Dolphin Quest Waikoloa (DQW) for their support. Specifically we thank Carla Buczyna (DQW), Michelle Campbell (DQW). We thank Brad Andrews (SeaWorld Parks and Entertainment) for his support of this project.

References

1.  O'Brien JK, Robeck TR (2006). Development of sperm sexing and associated assisted reproductive technology for sex pre-selection of captive bottlenose dolphins (Tursiops truncatus). Reproduction Fertility and Development 18:319-329.

2.  O'Brien JK, Steinman KJ, Robeck TR (2009). Application of sperm sorting and associated reproductive technology for wildlife management and conservation. Theriogenology 71:98-107.

3.  Robeck TR, Steinman KJ, Yoshioka M, Jensen E, O'Brien JK, Katsumata E, Gili C, McBain JF, Sweeney J, Monfort SL (2005). Estrous cycle characterization and artificial insemination using frozen-thawed spermatozoa in the bottlenose dolphin (Tursiops truncatus). Reproduction 129:659-674.

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