Kadaknath chicken at a poultry farm in Dantewada, India. Credit: Sanchit Khanna/Hindustan Times via Getty Images

An Indian research team has for the first time successfully transferred primordial chicken germ cells into a duck embryo which developed into a duckling that stored chicken germ cells in its reproductive organs1.

The germ cells, which form ova or sperm cells, can be harvested from the resultant duckling to breed rare and endangered chicken varieties.

“Our research offers a way to biobank chicken germplasms in ducks which are immune to various diseases, including bird flu,” said Adnan Naim, one of the researchers at the KIIT University in Bhubaneswar. The successful experiment is a step towards conserving chicken germplasm.

India does not have technology to conserve chicken germplasms using primordial germ cells. Indian poultry farmers adopt an expensive strategy of rearing and maintaining a reserve of 500 to 1,000 captive chickens, which are at risk of dying from avian flu.

Germ cell culture to cryopreservation

Naim, Suryakant Mishra at the Indian Council of Agricultural Research-Directorate of Poultry Research in Bhubaneswar and Debasis Nayak at the Indian Institute of Technology in Indore developed methods to conserve chicken germplasm.

The researchers selected the Kadaknath chicken, an Indian breed with highly pigmented bones and muscles. The pigments in this chicken breed make it easier to track the transmission of germ cells chicken to chicken, or chicken to duck.

Once formed in the blood, the primordial germ cells migrate to the reproductive organs in growing embryos. The scientists collected the cells from the blood of a 2.5-day-old embryo and the reproductive organs of a 6.5-day-old embryo of the Kadaknath chicken. The germ cells were grown in a nutrient-rich cell culture conditioned with buffalo rat liver (BRL) cells. After three months, the BRL cells increased the proliferation of germ cells by one and a half times.

Kadaknath primordial germ cells on buffalo rat liver feeder cells. Credit: Adnan Naim, Debasis Nayak

The team isolated the proliferated germ cells from the culture and identified the expression of germ-cell-specific genes using a newly patented procedure to preserve the germ cells in liquid nitrogen with specific cryoprotectants.

“Germ cells can be stored in vials containing liquid nitrogen for an indefinite period,” said Naim. The germ cells’ potential to eventually differentiate into ova or sperm cells remained unaltered during the storage period, he added.

The researchers said their technology has the potential to reduce the cost of maintenance of chicken germplasm by 50% to 60%.

“Cryopreservation of primordial germ cells from the Kadaknath chicken can ameliorate risk of infectious diseases such as avian flu, because the germ-cells-mediated technology can conserve both male and female fertility,” says Yoshiaki Nakamura, an expert on reproductive biology, at the Hiroshima University in Japan, who was not involved with the research.

Chimaera: Hosting cells of two different breeds

The team stained the cultured and frozen Kadaknath germ cells with a light-emitting dye and transferred them into 2.5-day-old embryos of the White Leghorn chicken to test whether they would colonise the reproductive organs of another chicken breed. After six days, they identified the dye-stained germ cells in the reproductive organs of the Leghorn embryos.

The Kadaknath germ cells can eventually form sperm cells or ova in the reproductive organs of the Leghorn embryos, called chimaera. A Leghorn embryo bearing Kadaknath sperm cells in its reproductive organs is called a male chimera. “A male chimera back-crossed with a female Kadaknath chicken can produce Kadaknath chickens,” Naim said. A female chimera is a Leghorn embryo hosting Kadaknath’s ovary. Back-crossing such a female chimera with a male Kadaknath chicken would also produce Kadaknath chicks.

“We can safely assume the possibility of successful restoration of indigenous chicken breeds using such techniques,” said T S Thiyagasundaram, an avian geneticist and consultant at the National Agricultural Higher Education Project of Indian Council of Agricultural Research.

Thiyagasundaram said the success rate of restoration will be higher if the donor and surrogate-recipient belong to the same species, such as chickens of two breeds.

Ducks: Surrogate hosts to chicken reproductive organs

The researchers transplanted the Kadaknath’s testes into the reproductive organs of ducklings of Khaki Campbell breed, which did not trigger any adverse immune responses. After eight weeks, 70% to 80% of the ducklings survived without immuno-suppressive drugs.

The researchers also demonstrated that the Kadaknath germ cells derived from blood and reproductive organs successfully colonised the reproductive organs of White Pekin duck embryos. The embryos, called chicken-duck germline chimera, hatched after four weeks2.

Ducks, with natural immunity to many diseases that afflict chickens, can provide a robust surrogacy system for chicken germ cells, said Mishra.

“The transplanted reproductive organs may remain alive and active in the recipient birds even long after the original donor birds are dead or extinct,” said Sunit Kumar Mukhopadhyay, an expert on animal diseases at the West Bengal University of Animal & Fishery Sciences in Kolkata.

The researchers said that the technology could prove useful to conserve germplasms of wild and endangered birds such as vultures. “We are optimistic that the primordial germ cells-based technologies can revolutionise biobanking for endangered birds,” Naim said.

Nakamura cautioned it was too early to guarantee success of this technology, as while primordial germ cells of a donor were found in the reproductive organs of a recipient bird, germ cells of a donor bird had not been observed to differentiate into functional reproductive cells such as ova.