Semen collection

Aquarium sand tiger sharks were manually restrained and a stream of oxygenated water created from bubbling oxygen gas was directed at the shark’s mouth using a submerged pump, and respiration rate was monitored throughout restraint [33]. In situ sand tiger sharks were sampled from South Carolina (April) and Delaware Bay (June–August) and restrained boat-side or transferred to a livewell aboard the vessel for examination. Restraint included dorsoventral rotation to induce tonic immobility (TI) [34, 35] for no longer than 30 min. Semen was collected using a sterile 16-inch 18Fr polyvinyl chloride catheter with a rounded, closed tip (Kendall/Covidien, Mansfield, MA) inserted through the urogenital papilla and into an ampulla (15–20 cm). Semen was extracted using gentle suction from a catheter tip syringe (Monoject, Kendall/Covidien, Medtronic, Minneapolis, MN) while slowly withdrawing the catheter through the length of the ampulla. Semen was transferred to sterile conical tubes (Thermo Fisher Scientific, Waltham, MA) after collection.

Care was taken to preserve catheter sterility before entry into the urogenital papilla, and once introduced into an ampulla, the catheter was not removed until semen collection was complete. Semen samples were maintained in the dark at 4 °C until analysis and assessed at ambient temperature (22–25 °C) within 24 h. To investigate the prevalence of potential pathogenic organisms that might be deleterious in an artificial insemination program, semen samples (n = 7 in situ, n = 4 aquarium) from sand tiger sharks were collected onto a sterile culturette (BBL CultureSwab Plus, Becton, Dickenson and Company, Sparks, MD) immediately after collection and shipped to the University of Georgia Veterinary Diagnostic Laboratories, Athens, GA, California Microbiological Reference Laboratory, Tustin CA, or IDEXX Laboratories, Inc, ME for aerobic culture.