N OCIETY' - the Society for Reproductive Biology

SEMEN COLLECTION, CHARACTERISATION AND CRYOPRESERVATION IN A MAGELLANICPENGUIN (Spheniscus magellanicus).J. K. O'Brien, D. A. Oehler, S. P. Malowski and T. L. RothCenter for Research ofEndangered Wildlife, Cincinnati Zoo and Botanical Garden, Cincinnati, Ohio 45220, USA.IntroductionSeven of the 17 penguin species in existence areendangered (1). A better understanding of avianreproduction and the application of assisted breedingtechniques such as semen preservation and artificialinsemination (AI) may become important for captivepropagation and long-term survival of these species.However, information on ejaculate characteristics andsemen preservation is not available for any penguinspecies. The Magellanic penguin is not endangered, butits reproductive success in the wild has been decliningduring the past decade (2). The objectives of thepresent study were to: (i) develop a cooperative methodfor collecting Magellanic penguin semen; (ii)characterise the ejaculate parameters and semenproduction during a breeding season; (iii) compareholding temperatures for short-term storage; and (iv)compare cryoprotectants for long-term storage.Materials & MethodsSemen was obtained from a hand-reared adult maleusing a cooperative collection method. Ejaculates wereassessed for volume, pH and osmolality. Sperm qualitywas assessed by evaluating motility and forwardprogression (sperm motility index, SMI), viability(Hoechst 33258) and morphology.For short-term storage (n=3 replicates), semen wasdiluted (1:3; semen:diluent) with Beltsville PoultrySemen Extender (BPSE; 3) within 10 min of collection.BPSE pH and osmolarity were adjusted to simulateMagellanic penguin semen pH (7.4 ± 0.5) andosmolarity (430 ± 10 mosm/kg). The diluted semen wasdivided into 2 aliquots and held at 4°C or 21°C. Spermquality assessments were made at 0, 1 and 3 h postdilution.For long-term storage (n=3 replicates), diluted (l:1)semen was further diluted (1:1) with BPSE containingdimethylsulfoxide (DMSO) or ethylene glycol (EG),and equilibrated for 15 min (final cryoprotectantconcentration: 8%). Straws were frozen on dry ice for 5min and transferred to liquid nitrogen. Semen wasthawed (n=2 straws/sample) by holding straws for 5 sinair and then by placing them in a 50°C waterbath for 10s. Data were analysed by analysis of variance, andmeans were compared using Students t-tests or LSDtests. P0.05) in spermcharacteristics between treatments for short-termstorage, although there appeared to be a slightadvantage to maintaining semen at 4°C. Semen samplesmaintained high numbers of viable (77.8 ± 5.4%) andmorphologically normal (67.9 ± 2.5%) sperm at 3 hours(data for both treatments combined). SMI and percentviability decreased (P0.05) ascryoprotectants for penguin spermatozoa (data notshown). Frozen-thawed semen maintained 68.5 ± 4.6%and 78.4 ± 3.2% of its pre-freeze SMI and viability,respectively (data pooled for cryoprotectant). Both SMIand viability remained relatively stable during the 3 hstorage interval post-thaw (Table 1). Similar to freshlycollected ejaculates, frozen-thawed semen exhibited anincrease in bent cells (P