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On
the left, Jennifer Nagashima, SCBI doctoral fellow, and on the right,
Nucharin Songsasen, SCBI scientist, with two of the puppies born using
IVF.
December 9, 2015
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.
"... dogs are weird," said Jennifer Nagashima, a doctoral fellow at the Smithsonian Conservation Biology Institute. "Dogs 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."
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.
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.
Finally, the team was tasked with returning the fertilized egg to the surrogate mother... 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.
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 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.”
pen Access
This lack of progress is remarkable given the critical role ART could play in conserving endangered canid species or eradicating heritable disease through gene-editing technologies—an approach that would also advance the dog as a biomedical model.
Over 350 heritable disorders/traits in dogs are homologous with human conditions, almost twice the number of any other species.
Here we report the first live births from in vitro fertilized embryos in the dog.
Adding to the practical significance, these embryos had also been cryopreserved.
Changes in handling of both gametes enabled this progress. The medium previously used to capacitate sperm excluded magnesium because it delayed spontaneous acrosome exocytosis.
We found that magnesium significantly enhanced sperm hyperactivation and ability to undergo physiologically-induced acrosome exocytosis, two functions essential to fertilize an egg.
Unlike other mammals, dogs ovulate a primary oocyte, which reaches metaphase II on Days 4–5 after the luteinizing hormone (LH) surge.
We found that only on Day 6 are oocytes consistently able to be fertilized.
In vitro fertilization of Day 6 oocytes with sperm capacitated in medium supplemented with magnesium resulted in high rates of embryo development (78.8%, n = 146).
Intra-oviductal transfer of nineteen cryopreserved, in vitro fertilization (IVF)-derived embryos resulted in seven live, healthy puppies.
Development of IVF enables modern genetic approaches to be applied more efficiently in dogs, and for gamete rescue to conserve endangered canid species.
(B) Ultrasound image of a normally-developing embryo imaged Day 29 from Transfer #5.
(C) 7 healthy puppies were born by planned Caesarian section.
(D) Normally developing beagle puppy at 3 weeks of age.
In the current study we determined that:
The first test tube puppies have been born:
http://bit.ly/1Y2PjYA
http://bit.ly/1Y2PjYA
The First Test Tube Puppies Have Been Born
After decades of research and attempted trials, it was the first time in-vitro fertilization (IVF) had been carried out successfully in a dog.
"... dogs are weird," said Jennifer Nagashima, a doctoral fellow at the Smithsonian Conservation Biology Institute. "Dogs 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."
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.”
Three of the seven puppies born using IVF.
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.
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.
Finally, the team was tasked with returning the fertilized egg to the surrogate mother... 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.
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 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.”
pen Access
Peer-reviewed
Research Article
Abstract
Development of assisted reproductive technologies (ART) in the dog has resisted progress for decades, due to their unique reproductive physiology.This lack of progress is remarkable given the critical role ART could play in conserving endangered canid species or eradicating heritable disease through gene-editing technologies—an approach that would also advance the dog as a biomedical model.
Over 350 heritable disorders/traits in dogs are homologous with human conditions, almost twice the number of any other species.
Here we report the first live births from in vitro fertilized embryos in the dog.
Adding to the practical significance, these embryos had also been cryopreserved.
Changes in handling of both gametes enabled this progress. The medium previously used to capacitate sperm excluded magnesium because it delayed spontaneous acrosome exocytosis.
We found that magnesium significantly enhanced sperm hyperactivation and ability to undergo physiologically-induced acrosome exocytosis, two functions essential to fertilize an egg.
Unlike other mammals, dogs ovulate a primary oocyte, which reaches metaphase II on Days 4–5 after the luteinizing hormone (LH) surge.
We found that only on Day 6 are oocytes consistently able to be fertilized.
In vitro fertilization of Day 6 oocytes with sperm capacitated in medium supplemented with magnesium resulted in high rates of embryo development (78.8%, n = 146).
Intra-oviductal transfer of nineteen cryopreserved, in vitro fertilization (IVF)-derived embryos resulted in seven live, healthy puppies.
Development of IVF enables modern genetic approaches to be applied more efficiently in dogs, and for gamete rescue to conserve endangered canid species.
Fig 3. Results of transfer of IVF embryos.
(A) Embryo stage, status, transfer location and the results of IVF-derived embryo transfers.(B) Ultrasound image of a normally-developing embryo imaged Day 29 from Transfer #5.
(C) 7 healthy puppies were born by planned Caesarian section.
(D) Normally developing beagle puppy at 3 weeks of age.
doi:10.1371/journal.pone.0143930.g003
1) magnesium had positive
effects supporting sperm hyperactivation and acrosome exocytosis, two
functions essential for fertilization competence;
2) Day 6 post-LH surge
was optimal for obtaining developmentally competent oocytes;
3) there
was no significant effect of progesterone on embryo production;
4) male
donor significantly influenced oocyte fertilization and embryo
development in the dog; and 5) it is possible to produce live young from
IVF-derived, frozen-thawed embryos transferred into the oviduct.
Generation of a successful protocol for IVF in the dog lays the
foundation for application of gene-editing technologies and also
provides a means to perform gamete rescue in endangered canid species.
Citation: Nagashima JB, Sylvester SR, Nelson JL, Cheong SH, Mukai C, Lambo C, et al. (2015) Live Births from Domestic Dog (Canis familiaris) Embryos Produced by In Vitro Fertilization. PLoS ONE 10(12):
e0143930.
doi:10.1371/journal.pone.0143930
Editor: Jason Glenn Knott, Michigan State University, UNITED STATES
Received: October 11, 2015; Accepted: November 9, 2015; Published: December 9, 2015
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication
Data Availability: All relevant data are within the paper.
Funding: This research was supported by the National Institutes of Health DP-EB016541 (A.J.T.), Cornell University’s Baker Institute for Animal Health (A.J.T.), the Atkinson Center for a Sustainable Future (A.J.T.), and the Smithsonian Institution Predoctoral Fellowship (J.B.N.).
Competing interests: The authors have declared that no competing interests exist.
Editor: Jason Glenn Knott, Michigan State University, UNITED STATES
Received: October 11, 2015; Accepted: November 9, 2015; Published: December 9, 2015
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication
Data Availability: All relevant data are within the paper.
Funding: This research was supported by the National Institutes of Health DP-EB016541 (A.J.T.), Cornell University’s Baker Institute for Animal Health (A.J.T.), the Atkinson Center for a Sustainable Future (A.J.T.), and the Smithsonian Institution Predoctoral Fellowship (J.B.N.).
Competing interests: The authors have declared that no competing interests exist.
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