Abstract

The development of artificial insemination in marine mammals has required a long-term commitment toward understanding the basic reproductive physiology of the species involved. In addition to the bottlenose dolphin, artificial insemination (AI) using cooled transported semen has recently been confirmed via ultrasonography in the killer whale. This successful conception represents the first marine mammal conceived by AI and it is the result of years of basic research into the reproductive biology of this species. As is necessary for the development of assisted reproductive technologies (ART) in any species, research efforts with the killer whale have been concentrated on reproductive anatomy and endocrinology, and sperm cryobiology. Anatomical studies reveal a similar placentation (diffuse, epitheliochorial) and uterine conformation (bicomuate with short body and elongated horns) among all delphinids examined thus far, cervical conformation, however, appears diverse. The killer whale cervical complex consists of two cervices in series. Each cervix is composed of longitudinal folds similar in structure to an equine cervix. The distance between the internal os of the distal cervix and the external os of the proximal cervix and its surrounding fornix form a space where an erroneously placed catheter can easily lodge. This double cervical confirmation combined with a 60 to 70 cm vagina, necessitates the use of an endoscope for intrauterine inseminations.

The bottlenose dolphin is the only cetacean where semen cryopreservation studies have been performed. The publications concerning bottlenose dolphin cryopreservation are few and offer little information concerning many of the basic bottlenose dolphin sperm freezing characteristics. Preliminary research with killer whale spermatozoa indicate that when extended with egg yolk citrate, an egg yolk based extender (egg yolk citrate, 20% egg yolk, 3.2% Na citrate dihydrate, titrated to pH 6.8 using 5% citric acid monohydrate solution) it can be held for at least two days at 4°C while maintaining a total motility (TM) of up to 95%, progressive motility (PM) of 95% and a kinetic rating (KR) of 4.0. Killer whale sperm cryopreservation studies have found post-thaw TM ranging from 50 to 60%, PPM 95% and KR of 3.5 to 4.0 using the commercial bovine extender Biladyl®, (Minitube of America, Verona, WI 930187, USA) in 7% glycerol, frozen in 0.5 cc straws, at an approximate freezing rate of -11.4°C per minute for 10 minutes prior to a liquid nitrogen plunge. The ability to cool killer whale semen while retaining its fertility was critical for the successful application of AI in this species. Once proven successful, the use of cryopreserved semen for AI will allow for greater application of this technology.

The female killer whale was the first captive cetacean to be trained for voluntary urine collection. This capability has led to an extensive understanding of their hormonal profiles during estrus. Significant findings include the confirmation that estrogen conjugates provide the most accurate marker for predicting ovulation. With this information, and in conjunction with sporadic application of the ultrasonic ovarian evaluation technique perfected in bottlenose dolphins¹, it has been possible to determine an appropriate time for insemination with cooled semen.

Despite these recent successes, little information has been collected concerning reproductive endocrinology of other cetacean species, including the bottlenose dolphin. Hormonal fluctuations that occur and are responsible for follicular development and ovulation have not been described. Current estrus synchronization methods should be refined and other combination hormonal treatments should be tested. Collection of semen has proven difficult to apply to a range animals and species. Methodologies for training these behaviors need to be compiled and shared with managers. Before AI can be developed in other cetacean species, these basic reproductive biological parameters must be understood. In addition, information obtained from this early research must be applied to a large number of animals within each species before any real benefit to captive management will be observed.

Acknowledgements

I would like to thank Drs. Scott Gearhart, Tom Reidarson, and Steve Monfort of SeaWorld Orlando, SeaWorld San Diego and the Conservation and Research Center, National Zoological Park, respectively, for their assistance. I also thank Karen Steinman of CRES, and the animal training and Laboratory staff at SeaWorld Orlando, San Antonio and San Diego.

References

1.  Brook, F., 2000, Sonographic testicular and ovarian assessment in the bottlenose dolphin, Tursiops truncatus aduncus, in Report from the bottlenose dolphin breeding workshop, Duffield,D,A. and Robeck,T.R., Eds., American Zoological Association Marine Mammal Taxon Advisory Group, Silver Springs, MD, 207-222.