Chicks Produced From Artificial Insemination in the Magellanic Penguin (Spheniscus magellanicus) Using Chilled Semen
IAAAM 2014
Justine K. O'Brien1; Justin E. Brackett2; Lauren R. DuBois2; Todd L. Schmitt2; Hendrik H. Nollens2; Karen J. Steinman1; Jean M. Dubach3; Todd R. Robeck1
1SeaWorld Parks and Entertainment Inc., SeaWorld and Busch Gardens Reproductive Research Center, San Diego, CA, USA; 2SeaWorld San Diego, SeaWorld Parks and Entertainment Inc., San Diego, CA, USA; 3Loyola University Medical Center, Maywood, IL, USA


Zoological penguin populations represent an important biological resource that can facilitate an extensive understanding of a species' biology. This knowledge is a prerequisite for developing in situ population management plans designed to address impacts of naturally occurring or anthropogenic stressors on population survival. Assisted reproductive technologies including artificial insemination (AI) and semen preservation can potentially be used to improve propagation rates and genetic diversity of in situ or ex situ penguin populations in conjunction with natural breeding. The objectives of this research with the Magellanic penguin (Spheniscus magellanicus) were to: (i) examine the fertility of chilled-stored semen after cloacal AI; (ii) determine semen and sperm parameters of conceptive inseminations and; (iii) characterize profiles of plasma hormone and biochemical parameters relative to oviposition. At the onset of the 2013 breeding season, four females were separated from their paired male by transparent fencing material, and remained in their cordoned nest site until oviposition was completed. Cloacal inseminations under anesthesia and blood collection for biochemistry and hormone analyses were performed 1 day after pair separation, and once weekly thereafter until completion of laying. At least two additional weekly blood samples were collected after the final oviposition. Semen was collected up to 20 h prior to insemination from six males using a cooperative method,1 diluted 1:1 (v/v) with Beltsville Poultry Semen Extender containing 10 µg/ml gentamycin sulfate (supplemented with LincoSpectin; Pharmacia & Upjohn, Kalamazoo, MI, USA), and stored at 5°C for up to 20 h prior to insemination. The semen donor was different for each week of insemination to allow determination of the sire and duration of in vitro and in vivo sperm storage relative to AI. All eggs were removed for artificial incubation and replaced with dummy eggs between 0–30 days post-laying depending on pair reproductive history. Seven eggs were laid and all were fertile based on candling observations and post-hatch genetic analysis using nine microsatellite loci.2,3 An average of 3.3 ± 1.0 inseminations were performed in each bird prior to the final oviposition (17.5 ± 7.0 day separation interval). Fertile eggs were laid 5–11 days after AI with semen that was chilled 3–20 h prior to AI. For conceptive inseminations, 2.2–389.9 x 106 motile spermatozoa were used, and samples evaluated within 2 h prior to AI exhibited 89.2 ± 7.5% total motility, 68.2 ± 14.3% progressive motility, 62.0 ± 11.2 µm/s average pathway velocity and 68.8 ± 29.5% normal morphology. Four of the seven fertile eggs hatched and genetic analyses confirmed that all four chicks were sired by AI males. Plasma concentrations of progesterone, testosterone, estradiol, triglycerides, iron, calcium and phosphorus were elevated (p < 0.05) prior to oviposition compared with post-laying values. Results from this study demonstrate for the first time in any penguin species that chicks can be produced after AI, and that high fertility rates are achieved up to 11 days after insemination of chilled semen.


The authors thank the aviculture (Penguin Encounter), curatorial and veterinary staff at SeaWorld San Diego (SWSD) for their support and assistance with semen collection, blood collection, anesthesia and egg candling procedures. SeaWorld and Busch Gardens Reproductive Research Center staffs are thanked for technical support, as are SWSD's Animal Care Lab staffs for plasma biochemical analyses. The authors are also grateful to Dr. Terry Nett and staff at Colorado State University for reproductive hormone analyses, and to Mr. Brad Andrews of SeaWorld Parks and Entertainment for support of this research.

Literature Cited

1.  O'Brien JK, Oehler DA, Malowski SP, Roth TL. Semen collection, characterization and cryopreservation in a Magellanic penguin (Spheniscus magellanicus). Zoo Biol. 1999;18:199–214.

2.  Schlosser JA, Garner TWJ, Dubach JM, McElligott AG. Characterization of microsatellite loci in Humboldt penguin (Spheniscus humboldti) and cross-amplification in other penguin species. Mol Ecol Notes. 2003;3:62–64.

3.  Schlosser JA, Dubach JM, Garner TWJ, Araya B, Bernal M, Simeone A, Smith KA, Wallace RS. Evidence for gene flow differs from observed dispersal patterns in the Humboldt penguin, Spheniscus humboldti. Conserv Genet. 2009;10:839–849.


Speaker Information
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Justine K. O'Brien
SeaWorld Parks and Entertainment Inc.
SeaWorld and Busch Gardens Reproductive Research Center
San Diego, CA, USA

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