Earlier this summer, a pregnant beagle gave birth to seven adorable puppies—three females and four males. Five of the puppies were sired by another Beagle, but the two remaining puppies were sired by a Cocker Spaniel. And the mother? Another dog entirely.
After decades of research and attempted trials, it was the first time in-vitro fertilization (IVF) had been carried out successfully in a dog.
"I like to say that dogs are weird," said Jennifer Nagashima, a doctoral fellow at the Smithsonian Conservation Biology Institute. "They have some unique quirks to their reproduction."
Nagashima is the lead author of a report published on Wednesday in the open access journal PLOS ONE which documents the first live canine births from in vitro fertilized embryos using cryopreserved eggs and sperm. Researchers from the Smithsonian and Cornell University implanted nineteen embryos that had been fertilized using their technique, which resulted in the birth of "seven live, healthy puppies," according to the study.
Their research could help us not only better understand the hundreds of genetic traits and disorders that humans share with dogs, but to remove harmful traits that have been passed down through generations of breeding.
"A lot of the gold standard work for the last 30 years or so has tried to build on [1970s research], rather than looking back and evaluating our our basic assumptions," Nagashima explained in an interview. "So when we were approaching IVF, and trying to solve the problems of IVF in the domestic dog, we wanted to have optimal sperm, having optimal mature eggs, and then the culture conditions for the IVF or embryo."
Prior studies used immature eggs—often collected from ovarian follicles, "because of difficulty in obtaining oocytes after ovulation," according to the paper—and then attempted to mature them in a petri dish, Nagashima explained. However, these eggs proved ineffective at producing successful embryos. Her team suspected the quality of the eggs might be an issue—and indeed, found that eggs collected six days after ovulation are "consistently able to be fertilized."
Though it is more complicated to obtain eggs in this way—there's a fat pad covering the oviduct that makes it difficult to access—Nagashima's team found that, by leaving the eggs to mature for an extra day before removing them from the oviduct, the rate at which eggs could be fertilized was much higher than was found in previous studies.
"In those studies I think the highest success rate with regard to embryo production was around 30 to 35 percent, and our study was regularly getting about 70 to 75 embryo production," she said.
This higher success rate was also due, in part, to how Nagashima's team prepared their sperm. In previous research, magnesium had been omitted when preparing sperm, because researchers found it delayed a process called "acrosome exocytosis," which helps the sperm penetrate the egg. However, the new research found this delay "isn't necessarily a bad thing," Nagashima said. The addition of magnesium actually greatly increases the sperm's movement, or hyperactivated motility, just prior to fertilization, making the rate of fertilization much higher than without, despite the delay.
Finally, the team was tasked with returning the fertilized egg to the surrogate mother. In four out of five dogs studied, Nagashima's team deposited the embryos in the uterine horn—a part of the canine reproductive system which the embryo reaches later in its development. These eggs did not result in any births. However, for the fifth and final dog, nineteen embryos were deposited back into the surrogate Beagle's oviduct, which, according to Nagashima, was "the more physiologically appropriate location" for an embryo of a young age.
In this dog, the procedure worked.
According to Nucharin Songsasen, one of the paper's co-authors, their results could have implications not merely for dogs, but research on humans as well.
Songsasen mentioned that the IVF procedure they've developed could help maintain genetic diversity in zoos, where captive animals may be disinterested or unable to mate with one another. It could also mean that working dogs could have their genetic material preserved before spaying, and still breed later on, to preserve dogs with especially desirable traits. Coupled with the gene-editing technique CRISPR/Cas9, IVF could even make it possible to eradicate breed-specific ailments such as hip dysplasia dog or urinary stones, for which some breeds are especially predisposed.
"Dogs get a lot of genetic diseases and cancers, and a lot of of them are similar to humans," Nagashima explained. "So if we can really understand how these diseases work in dogs, then that would be the first step in eradicating them."