Semen stored since 1968 in a laboratory in Sydney has been defrosted and successfully used to impregnate 34 Merino ewes, with the resulting live birth rate as high sperm frozen for just 12 months.
“This demonstrates the clear viability of long-term frozen storage of semen. The results show that fertility is maintained despite 50 years of frozen storage in liquid nitrogen,” said Associate Professor Simon de Graaf from the Sydney Institute of Agriculture and School of Life and Environmental Sciences at the University of Sydney.
“The lambs appear to display the body wrinkle that was common in Merinos in the middle of last century, a feature originally selected to maximise skin surface area and wool yields. That style of Merino has since largely fallen from favour as the folds led to difficulties in shearing and increased risk of fly strike,” Associate Professor de Graaf said.
His colleague on this project, Dr Jessica Rickard, said: “We believe this is the oldest viable stored semen of any species in the world and definitely the oldest sperm used to produce offspring.”
Associate Professor de Graaf said that it was the reproductive biology and genetic aspects of these as-yet unpublished findings that were of most interest to him.
“We can now look at the genetic progress made by the wool industry over past 50 years of selective breeding. In that time, we’ve been trying to make better, more productive sheep,” he said. “This gives us a resource to benchmark and compare.”
Strong tradition in animal science
Dr Rickard is a post-doctoral McCaughey Research Fellow in the Sydney Institute of Agriculture. She is continuing the strong animal reproduction research tradition in veterinary and biological sciences at the University of Sydney through her work in the Animal Reproduction Group.
Dr Rickard did the original work to determine if the stored semen was viable for artificial insemination. This involved thawing the semen, which is stored as small pellets in large vats of liquid nitrogen at -196 degrees. She and her colleagues then undertook in vitro tests on the sperm quality to determine the motility, velocity, viability and DNA integrity of the 50-year-old sperm.
“What is amazing about this result is we found no difference between sperm frozen for 50 years and sperm frozen for a year,” Dr Rickard said.
Out of 56 ewes inseminated, 34 were successfully impregnated. This compares to recently frozen semen from 19 sires used to inseminate 1048 ewes, of which 618 were successfully impregnated. This gives a pregnancy rate of 61 percent for the 50-year-old semen against 59 percent for recently frozen sperm, a statistically equivalent rate.
The results are part of a long tradition at the University of Sydney in animal reproduction research. The original semen samples were donated in the 1960s from sires owned by the Walker family. Those samples, frozen in 1968 by Dr Steven Salamon, came from four rams owned by the Walkers on their then property at Ledgworth.
The rams were of Ledgworth, Merryville and Boonoke genetics, famous names in the wool industry. The Boonoke ram (known as ‘Sir Freddie’) was born in 1959 and sold to Ledgworth in 1961 for 345 guineas. The Merryville ram, born in 1963 was bought by Ledgworth in 1965 for 1000 guineas. The other two rams are progeny of the Boonoke and Merryville rams, born in 1963 and 1965, respectively.
The Walkers now run 8000 sheep at ‘Woolaroo’, at Yass Plains, and maintain a close and proud relationship with the animal breeding program at the University of Sydney.
Peter Walker said: “Me and my family have been working with the University of Sydney and the veterinary unit since 1962. An association of more than 60 years – it’s a long time.”
Mr Walker said after developing his relationship with the university in the early 1960s, the Searle’s drug company in the US, which released the contraceptive pill, used his Merinos for testing the pill.
“The Merino sheep has played important role that has flowed through to human reproduction,” he said. “Not only on the pill but also work from Neil Moore and Roger Bilton on freezing embryos.”
Mr Walker said his cooperation with the University has also involved hosting undergraduate Veterinary Science students. “Until a couple of years ago, I’ve been taking students every year since 1962.”
He praised the early role of Dr Steve Salamon. “He came up here for 20 consecutive years. His story is unbelieveable,” Mr Walker said.
“Who knew that 50 years ago, that the semen they used in the recent trial would have a higher fertility. Steve Salamon was an absolute perfectionist. And it wasn’t just for the professionals; he trained local farm workers with skills to this standard. It’s just a fantastic legacy.”
The research was undertaken in part courtesy of a grant from Australian Wool Innovation.