For the first time, a litter of puppies was born by in vitro fertilization, thanks to work by Cornell University researchers. The breakthrough, described in a study to be published online Dec. 9 in the journal Public Library of Science ONE, opens the door for conserving¹ endangered canid species, using gene-editing technologies to eradicate² heritable diseases in dogs and for study of genetic³ diseases. Canines⁴ share more than 350 similar heritable disorders⁵ and traits with humans, almost twice the number as any other species.

 

Nineteen embryos⁷ were transferred to the host female dog, who gave birth to seven healthy puppies, two from a beagle mother and a cocker spaniel father, and five from two pairings of beagle fathers and mothers.

 

"Since the mid-1970s, people have been trying to do this in a dog and have been unsuccessful," said Alex Travis, associate professor of reproductive biology in the Baker⁸ Institute for Animal Health in Cornell's College of Veterinary Medicine. 

 

Jennifer Nagashima, a graduate student in Travis' lab and the first to enroll⁹ in the Joint¹⁰ Graduate Training Program between the Smithsonian Conservation Biology Institute and Cornell's Atkinson Center for a Sustainable Future, is the paper's first author.

 

For successful in vitro fertilization, researchers must fertilize¹¹ a mature egg with a sperm¹² in a lab, to produce an embryo⁶. They must then return the embryo into a host female at the right time in her reproductive cycle.

 

The first challenge was to collect mature eggs from the female oviduct. The researchers first tried to use eggs that were in the same stage of cell maturation as other animals, but since dogs' reproductive cycles differ from other mammals, those eggs failed to fertilize. Through experimentation¹³, Nagashima and colleagues found if they left the egg in the oviduct one more day, the eggs reached a stage where fertilization was greatly improved.

 

The second challenge was that the female tract¹⁴ prepares sperm for fertilization, requiring researchers to simulate those conditions in the lab. Nagashima and Skylar Sylveste, found that by adding magnesium¹⁵ to the cell culture, it properly prepared the sperm.

 

"We made those two changes, and now we achieve success in fertilization rates at 80 to 90 percent," Travis said.

 

The final challenge for the researchers was freezing the embryos. Travis and colleagues delivered Klondike, the first puppy born from a frozen embryo in the Western Hemisphere in 2013. Freezing the embryos allowed the researchers to insert them into the recipient's oviducts (called Fallopian tubes in humans) at the right time in her reproductive cycle, which occurs only once or twice a year.

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