Abstract

Ultrasound has been used to estimate fecundity in both viviparous and oviparous species, negating the need for lethal sampling, which is problematic for those of conservation concern. While widely used to monitor pregnancy in viviparous species, ultrasonography is underutilized as a tool to characterize embryonic development in oviparous species.^1-4 Currently, a multi-institutional effort is underway to re-wild the endangered zebra shark (Stegostoma tigrinum) to locations where this species has been previously extirpated by leveraging aquarium collections as a source of brood stock.⁵ By this model, eggs from genetically appropriate parents are being shipped to Indonesia, where developing embryos will be hatched and reared prior to release. However, while zebra sharks are fecund, a large percentage of yolked eggs degrade or spoil within the first few weeks post-oviposition.^6,7 Therefore, ultrasonography represents a potential tool that could be used to both accurately distinguish fertile from non-fertile eggs and to diagnose changes in early embryonic development predictive of poor outcomes. The objectives of the current study were to use ultrasonography to assess egg fertility, monitor early embryonic development, and identify morphological indicators that may be predictive of early embryonic mortality.

Freshly laid eggs from four female zebra sharks were collected by aquarists daily from two enclosures at Aquarium of the Pacific. Eggs were tagged with a unique identification number, and date of oviposition was recorded. Eggs were incubated undisturbed for two or three weeks at 23.8–25°C and then underwent weekly exams via ultrasound to monitor embryo development. Eggs were evaluated for yolk integrity, egg case fluid homogeneity, and embryo morphology. In particular, embryo size, viability, appearance of developmental features (e.g., external gills, heartbeat, etc.), embryo movement (active, slow, or none), and morphological abnormalities were recorded weekly.

A total of 103 yolked eggs with embryo development were confirmed at first examination (mean 30 days) and were monitored over the course of 592 days. Only one embryo survived to hatch and was confirmed parthenogenic as were all other embryos able to be collected perimortem before autolysis. Abnormalities were observed in the majority of samples (n=69, 67%) as early as 16 days to 93 days post-oviposition. Common abnormalities included bent or curled tails, anechoic fluid pockets at the attachment of the yolk stalk to the yolk, and decreased movement. Mean lifespan was longer in embryos with an observed abnormality (61±20 days) than in embryos where no abnormality was visualized (43±13 days), likely due to embryos perishing before abnormalities could be detected. In the early stages of development, growth averaged 0.06 mm/day and doubled to as much as 0.13 mm/day for embryos that survived to at least 70 days post-oviposition. Overall, lifespan ranged from 27 days to 116 days (median 50 days), not including the hatched individual. Ultrasonography was found to be an effective and noninvasive method to determine egg fertility, identify embryos with developmental abnormalities, and track growth rates. Future work could employ ultrasonography to screen embryos prior to shipment for conservation-based projects or estimate hatch dates based upon embryo development when oviposition date is unknown.

Acknowledgments

Authors would like to thank Aquarium of the Pacific husbandry staff for contributions to this work.

*Presenting author

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