Abstract

Artificial insemination (AI) has been available for the genetic management of domestic animals for nearly 50 years. Despite widespread application in domestic animals, AI has only been successfully developed in a handful of exotic species. Successful development of AI techniques requires a thorough understanding of basic reproductive physiological parameters, including endocrinology, ovulation detection, anatomical description of the reproductive tract, and the development of sperm cryopreservation techniques. Research success is facilitated when biologic samples, including blood, urine, saliva, feces, and semen can be collected on a frequent basis. In fact, the failure to develop routine management, handling and sampling strategies has precluded the widespread application of AI techniques in wildlife species. Fortunately, marine mammals can be readily trained to provide biological specimens, and this has accelerated the development of AI in the killer whale, Orcinus orca, the bottlenose dolphin,¹ Tursiops truncatus, and most recently, the Pacific white-sided dolphin (PWSD), Lagenorhynchus obliquidens.

For this study, nine females and one male PWSD located at three facilities, SeaWorld of Texas, San Antonio, Texas USA (four females), John G. Shedd Aquarium, Chicago, Illinois USA (five females) and Kamogawa SeaWorld, Kamogawa, Chiba, Japan (one male) were used. All females were trained for voluntary urine collection, and daily samples were collected from July 1, 2000 to November 30, 2000, and again from July 1, 2002 to December 30, 2002. Urinary estrogen conjugates were measured by single antibody, direct enzyme immunoassay (EIA). The male was trained for voluntary semen collection. Once collected, semen was evaluated for total motility, percent progressive motility, and progression rate (subjective 0 to 5 rating). The semen was then extended 1:1 in egg-yolk citrate extender (3 percent sodium citrate, 20 percent egg yolk, q.s. distilled water) and cooled (4°C) for one hour. Next, semen/extender mix was equilibrated in an ice bath (2°C) for one hour. Egg-yolk citrate extender with 14 percent glycerol (pre-cooled to 2°C) was added 1:1 to extended semen to achieve a final glycerol concentration of 7 percent. Semen was immediately placed in 0.5 cc straws, incubated for one hour, then placed in liquid nitrogen vapor (2 cm above liquid) for 10 minutes prior to plunging in liquid nitrogen. Straws were stored in a standard cryotank until importation into the U.S. via a dry shipper (MVE Cryogenics, New Prague, MN 56071 USA). For estrus synchronization, all animals were administered of altrenogest (Regu-Mate®, Intervet Inc., Millsboro, DE 19966 USA) for 30 days (1 ml per 50 kg body weight, p.o., s.i.d.). Ovaries were evaluated by transabdominal ultrasound and serum drawn for progesterone determination on days 0 (being the first day of altrenogest administration), 15, 30, and 40. After day 40, ovaries were examined by ultrasound every three days until a follicle > 1.0 cm was observed, after which ultrasound examinations were conducted daily. Animals with significant follicular activity and rising urinary estrogen conjugates were targeted for AI.

For the AI, urinary estrogen was evaluated twice daily until an estrogen peak was observed in conjunction with a presumptive pre-ovulatory follicle. After this point, viable candidates were inseminated intrauterine using a 150-cm-long, 8.5 e.d. fiberoptic flexible gastroscope (Karl Storz Veterinary Endoscopy America, Santa Barbara, CA 93117 USA) until ovulation. After inseminations, progesterone concentrations were determined and serum was collected weekly for four weeks, and then bi-weekly for the subsequent two months. Animals with continually elevated progesterone were confirmed pregnant by ultrasound at eight weeks. During the first season (2001), none of the seven altrenogest-treated animals ovulated after two rounds of treatment. One (SWT) of the two animals not placed on altrenogest cycled naturally and was inseminated intra-cervically, but did not conceive. During the next season (2002), a single SWT female (one of five) ovulated after altrenogest treatment, was inseminated intra-cervically, but failed to conceive. This female subsequently ovulated naturally (27 days later), was inseminated intra-uterinely, and confirmed pregnant by ultrasound (Table 1 & 2). The three SWT females were re-treated with altrenogest, but none ovulated after cessation of hormone therapy (Table 1). For the animals at JGSA, two of five animals ovulated during the first altrenogest treatment interval; however, none were inseminated. One of the two ovulatory animals had a retained corpus luteum. The remaining four animals were re-treated with altrenogest for 30 days. Two of these females ovulated, were inseminated intrauterine, and confirmed pregnant by ultrasound (Table 1 & 2). This is the first time that AI has been used successfully in a Pacific white-sided dolphin. More significantly, we have demonstrated that frozen-thawed sperm (imported from Japan) can be used to augment the genetic diversity of closed Pacific white-sided dolphin populations.

Table 1. Synchronization results.

a. POFS = Preovulatory follicle size; b. Ovulation was missed.

Table 2. Artificial insemination trials.

a. Total number of inseminations for each ovulation; b. Total number of progressively motile sperm per insemination; c. Progressive motility rating (0 to 5, with zero = no motility and 5 = rapid forward movement); d. No Conception; e. Post Ovulatory; f. Before Ovulation; g. Utero-Tubo Junction; h. Conception; i. Intrauterine.

Acknowledgements

We thank Doug Acton (SWT), Britt Posey (SWT), Ken Ramirez (JGSA), Satoshi Inoue (KSW) for all of their efforts in conditioning the animals for sample collection. We thank Dr. Etsuko Katsumata (KSW) for her support. We also thank the animal care, training, and laboratory staffs at each facility for their assistance. Finally, we thank Sam Dover for the endoscope used during the AI procedures.

References

1.  Robeck TR, Atkinson S, Brook F. 2001. Chap 11: Reproduction. In: CRC Handbook in Marine Mammal Medicine, Second edition. Dierauf, L., Gulland, F. (eds). CRC Press, Boca Raton, Fl Pp. 193-236.