An embryo can appear perfectly preserved after freezing. According to new research, the real damage may not occur until the moment it begins to thaw.
Embryo vitrification has become a cornerstone of modern cattle reproduction, helping producers preserve valuable genetics, expand embryo transfer programs and accelerate genetic progress through in vitro fertilization (IVF). Yet despite decades of refinement, pregnancy rates following embryo transfer remain inconsistent.
New research suggests one reason may be hiding in plain sight.
Scientists from Cornell University, Dr. Jingyue Ellie Duan and Dr. Robert E. Thorne, have found that the greatest threat to embryo survival may not occur during freezing, as many have long assumed. Instead, significant ice formation may occur during warming, damaging embryos that appeared successfully vitrified during cooling.
The findings challenge a long-held assumption in cryopreservation and could lead to improved methods for preserving embryos, oocytes and other reproductive tissues.
What Is Vitrification and How Does It Protect Bovine Embryos?
The goal of vitrification is simple: cool an embryo rapidly enough that water does not form damaging ice crystals. Instead of freezing into crystalline ice, the embryo enters a glass-like state that protects cells during storage in liquid nitrogen.
Because ice crystals can damage cellular structures, vitrification has become the preferred method for preserving embryos used in IVF and embryo transfer programs.
For years, researchers focused primarily on preventing ice formation during cooling. Previous work, however, hinted that warming rates might play an equally important role.
“The question then was: Are cryopreservation agent concentrations, cooling rates, and warming rates in current cryopreservation practice adequate to prevent formation of significant ice during both cooling and warming/thawing?” Thorne says.
The question emerged from earlier work showing that the warming rates required to prevent ice formation are often dramatically higher than the cooling rates needed to achieve vitrification. In some cases, critical warming rates were found to be 100 to 1,000 times greater than critical cooling rates. That raised a troubling possibility: embryos could survive the freezing process without forming ice, only to sustain damage during warming.
To answer that question, the team used time-resolved X-ray diffraction and high-speed imaging to observe what was happening inside bovine embryos during vitrification and warming.
New Research Suggests Embryo Damage May Occur During Warming
“Using time resolved X-ray diffraction and high frame rate imaging we showed that while significant ice did not form during cooling, nearly all the free water inside bovine oocytes crystallized during warming. Most of the ice-related damage occurred during warming,” Thorne explains.
In other words, embryos that appeared successfully vitrified during freezing were still experiencing substantial ice formation during thawing.
Current vitrification protocols rely on cryoprotective agents, specialized handling techniques and rapid cooling to avoid ice crystal formation. The new findings suggest that even when those measures successfully prevent ice during cooling, embryos may remain vulnerable if warming occurs too slowly.
According to Thorne and Duan, the warming phase may provide enough time for ice crystals to form as embryos transition out of the vitrified state. Those crystals can damage cellular structures and reduce post-thaw viability.
This work builds on findings first reported by the team in 2024, strengthening evidence that warming-related ice formation may be a major source of cryopreservation injury.
Ice Formation During Thawing May Explain Poor IVF Outcomes
The findings could help explain why cryopreservation outcomes remain variable despite decades of improvements in embryo production and transfer technologies.
Perhaps most importantly, the research challenges a common assumption about vitrification success. An embryo that appears completely ice-free after cooling may still undergo substantial crystallization during warming. If embryos sustain damage during thawing, even after a successful vitrification process, that injury could contribute to reduced survival rates and less predictable pregnancy outcomes following embryo transfer.
The findings highlight warming as a potential target for improving reproductive success.
Improved Embryo Survival Could Benefit Dairy IVF Programs
The implications may be especially significant for dairy producers, who have become major users of IVF and embryo transfer technologies.
“The dairy cattle sector would benefit the most from improved embryo vitrification since it has extensive use of IVF and embryo transfer in elite dairy cattle,” Duan says.
Modern dairy breeding programs increasingly rely on IVF combined with sexed semen to accelerate genetic progress. Many operations also implant beef embryos into lower-tier dairy cows, producing beef-on-dairy calves that provide an additional revenue stream. Improving embryo survival after thawing could increase the efficiency and consistency of these programs while reducing losses associated with failed transfers.
The researchers also see opportunities to improve the preservation and distribution of valuable female genetics.
“These methods should be particularly useful for preserving and propagating elite female genetics, thereby driving livestock improvements,” Duan says.
While superior male genetics can be widely distributed through semen, preserving and transporting elite female genetics remains more challenging. Improved cryopreservation could help expand access to those genetics while improving reproductive efficiency.
Although dairy producers may be among the first beneficiaries, Duan and Thorne believe the same principles could improve outcomes in human fertility clinics and conservation programs focused on preserving endangered species.
How Protein Crystal Research Led to a Breakthrough in Embryo Cryopreservation
The project has roots outside reproductive biology and represents nearly two decades of work on cryobiology and cooling technology.
Beginning in the mid-2000s, the team studied cryocooling techniques used in protein crystallography. Their work led to the development of specialized sample holders and methods for dramatically increasing cooling rates using liquid nitrogen.
Because protein crystals and mammalian embryos share similar sizes and water content, researchers believed techniques developed for one field might benefit the other. In 2018, the team connected with the late Dr. Soon Hon Cheong of Cornell University’s College of Veterinary Medicine.
“He suggested using the bovine model because its lipid content and success rates are intermediate between humans and swine,” Thorne remembers.
Supported by UDSA funding awarded in 2022, the collaboration ultimately led to the studies reported in 2024 and 2026.
Faster Cooling and Warming Rates Could Reduce Cryopreservation Damage
These researchers are not only identifying a problem; they also believe they have tools that may help solve it.
“Using better sample holding technology can increase cooling and warming rates by a factor of 3–5. Better cryocooling technology can increase cooling rates by an additional factor of 10. These allow completely ice-free cryopreservation of oocytes and embryos,” Thorn explains.
Before those technologies can move into routine commercial use, additional work will be needed to adapt them to existing IVF and embryo transfer workflows. That includes refining sample holders, storage systems and tracking procedures for industry applications.
The team hopes to work with commercial users to determine how the technology can be integrated into real-world reproductive programs.
What Veterinarians Should Know About Embryo Vitrification Research
This study highlights an important shift in thinking. Rather than focusing exclusively on what happens during freezing, researchers are increasingly asking what happens during warming.
Thorne and Duan’s conclusion is direct:
“Using current industry-standard vitrification solutions, cooling methods and warming methods, ice always forms in your oocytes and embryos during warming, even when they appear ice-free after cooling. This ice is the main cause of sub-optimal cryopreservation outcomes.”
Eliminating ice formation may also help researchers better understand other causes of cryopreservation injury, including protein and lipid damage.
If future work confirms these findings, improving warming methods could become one of the next major advances in embryo transfer and IVF technology. Better cryopreservation could help producers preserve valuable genetics more effectively, improve embryo survival and increase the consistency of reproductive outcomes across the cattle industry.
