In the largest-ever study of artificial insemination in sharks, scientists overnighted the animals' semen to U.S. aquariums across state borders and successfully impregnated 20 females that subsequently birthed 97 pups, including some from rare asexual "virgin births."

The researchers behind the work, published Thursday in Scientific Reports, advocate for artificial insemination because aquariums often don't have enough sharks within a species to conduct traditional breeding practices. That limits genetic variation and lowers the probability of mating — crucial variables for aquariums because such institutions are integral in furthering marine research.

For instance, parthenogenesis, which refers to asexual reproduction or "virgin birth" — a rare phenomenon observed in the study — has exclusively been documented in an aquarium setting, with the exception of one instance.

"We probably wouldn't know parthenogenesis even existed if it wasn't for our aquarium colleagues," study author Kevin Feldheim, an adjunct assistant professor at Chicago's Field Museum of Natural History, told The Academic Times.

Cognizant of this treasure trove of information, Feldheim aimed to figure out how to safely maintain aquariums' shark populations in the new study. Led by Jennifer T. Wyffels, a postdoctoral researcher at the South‐East Zoo Alliance for Reproduction and Conservation, the team came up with cold-stored, long-distance artificial insemination as a solution.

"To help encourage and augment reproductive efforts of sharks in aquariums, artificial insemination might be one tool," Wyffels told The Academic Times. "Especially if you've got a population that's only females — they wouldn't even have the opportunity to mate."

Aquariums usually breed sharks across state lines when handlers transport sharks to other aquariums in hopes of finding the animals a partner — but that causes the animals stress. Previous studies have also attempted artificial insemination, Wyffels noted, but only with same-day shipped semen — limiting the travel radius.

"We used fresh and cold-stored semen and transferred it across the country and up and down the East Coast, moving it between institutions," Wyffels said. "That sort of proof of concept for the technique highlights the feasibility and efficacy of transporting [semen], rather than sharks, and is really a main finding."

Overnight shipping of semen would mean that sharks across the country could potentially mate with others of the same species, even from long distances, as the cold-stored semen was still viable after 24 or 48 hours with 28.1% and 7.1% success rates, respectively. Feldheim speculates that, in the future, this method can be extended to sharks in the wild, diversifying offspring even further.

"If we want to try to maximize the genetic diversity that we have in these institutions that just have, say, 40 individuals in aquariums, that's not the biggest gene pool," he explained. "Having the ability to collect sperm in the wild and then store that sperm … is an important step moving forward."

For proof of principle, along with a group of scientists working in designated aquariums for the study, Wyffels and Feldheim zeroed in on whitespotted bamboo sharks as models for the technique. 

Feldheim compares them to a larger type of shark, sand tigers — a species whose breeding patterns spurred the duo's initial interest in the project and one to which they hope to apply the method someday. While sand tigers are typically 6 to 8 feet long in aquariums, bamboo sharks are only about 3 feet long at most, making them easier to manipulate, Feldheim says.

Bamboo sharks also reproduce frequently; sand tigers, on the other hand, reproduce in an unpredictable manner so it's difficult to pinpoint fertility timing. Because the little model sharks are egg-layers, their offspring are also easier to test in laboratories, Wyffels said. Now that the study results are clear, she says the concept can be extended with minor adjustments to a more difficult species, including live bearers.

In this study, Wyffels first isolated the bamboo sharks' semen, then stored it in a cold storage box. She used standard overnight shipping to send it to various institutions nationwide — some on opposite ends of the country. 

Once the scientists at those institutions placed the semen into each female, the fish were allowed to swim freely in their tanks. When the sharks eventually laid eggs and the hatchlings grew a little bit, Feldheim stepped in. 

"I'm just making sure that the inseminations worked right," he explained. Feldheim turned to DNA tests to detect paternity, the same method used in humans. The entire process from insemination until confirmation of parentage took about six months. 

Upon comparing the genetic profiles of each offspring with those of the respective mother and father, the researcher confirmed that the baby sharks were a result of the artificial insemination and not an unknown prior natural mating. Unexpectedly, there were multiple instances of "virgin births."

Feldheim said that a few days after giving a virgin birth, one female shark gave birth again by sexual reproduction using the sperm she was inseminated with, which remained stored in her body. More than 70 days later, she spawned two more pups via virgin birth.

"We really have no idea what determines if an egg will develop from parthenogenesis or not," Wyffels said, "but in this study, we've shown a female is capable of switching very quickly — which is really exciting."

In addition to the benefit of genetic variation and increased mating patterns among sharks, Wyffels noted that artificial insemination has another advantage over traditional breeding. 

"Shark sex is pretty violent," she said. "Sharks don't have opposable thumbs or even hands, and instead, males use their mouths to grasp females during copulation."

"Having a bite wound from mating interactions is a part of their natural life history and not necessarily a bad thing," she continued, "but it's certainly another cost of reproduction [stressors] on the female."

The study, "Artificial insemination and parthenogenesis in the whitespotted bamboo shark Chiloscyllium plagiosum," published May 13 in Scientific Reports, was authored by Jennifer T. Wyffels, South-East Zoo Alliance for Reproduction & Conservation and University of Delaware; Lance M. Adams, Aquarium of the Pacific; Frank Bulman, Ripley's Aquarium of the Smokies; Ari Fustukjian, The Florida Aquarium and Loveland Living Planet Aquarium; Michael W. Hyatt, Adventure Aquarium and New York Aquarium, Wildlife Conservation Society; Kevin A. Feldheim, Field Museum of Natural History; and Linda M. Penfold, South-East Zoo Alliance for Reproduction & Conservation.