Cloning rescues rare cattle breed

Just over seven years ago, in August 1998, Dr David Wells of the Ruakua Research Centre in Hamilton announced the successful cloning of the last Enderby Island Cow, the last survivor of a the world's rarest cattle breed.

At the CSIRO Livestock Industries Horizons in Livestock Science conference in Surfers Paradise last month, Wells described the challenges involved in cloning cattle -- including transgenic cattle -- and the requirements for success.

Wells' Ruakua team was only the third in the world to clone a cow, after US and Japanese groups reported similar successes earlier in 1998.

Apparently related to the small, hardy cattle of the Shetland Islands, the breed was introduced to the sub-Antarctic island 320km south of New Zealand in 1894.

In 1991, as conservation measure, NZ Department of Conservation officers destroyed 47 cattle, after preserving semen samples from 16 bulls and oocytes from a number of cows. But efforts to produce embryos for transplantation into surrogate cows failed when the semen proved to be of poor quality, and the breed was presumed extinct.

In September 1992, two members of NZ's Rare Breeds Conservation Society found fresh hoofprints of two cattle on Enderby Island. Five months later, they captured the world's only surviving Enderby Island cow and her heifer calf and shipped them back to Massey University.

The calf subsequently died of unknown causes, leaving the cow, nicknamed Lady, as the only survivor of her breed in the world. Transferred to Ruakura, she received 35 embryo transplants before giving birth to a purebred bull, named Derby.

In a last effort to save the breed, Dr Wells cloned Lady, producing a heifer calf, Elsie (short for LC - 'Lady Clone'). Three more heifer clones were born, but two died. The entire herd -- Derby, Lady and the three surviving clones, now live on a were property near Christchurch, where two of the clones have since given natural birth to two heifer calves.

Wells told the Horizons conference the list of mammal species successfully cloned from somatic cells now includes cattle, sheep, mouse, rat, rabbit, mouflon, Banteng, monkey, horse, cat and dog.

Genetically modified somatic cells can be used to produce transgenic clones by somatic cell nuclear transfer -- the nuclear genome of the somatic cell is reprogrammed by its new environment in the evacuated oocyte.

Cloning was typically inefficient. Only 10 per cent of embryos from cloned cells produced viable offspring, compared with a success rate of 30 per cent for IVF, and 50 per cent for artificial insemination.

Wells said there was increasing evidence that faulty epigenetic programming -- a failure to reset the genes from the donor cell back to the embryonic state -- was the prime reason for the low success rate. For example, Dolly the sheep, the first cloned livestock species, had died prematurely because she was cloned from a mammary gland cell with a unique pattern of gene expression, markedly different from the embryonic pattern.

"The majority of clones show inappropriate patterns of chromatin modification, DNA methylation, and gene expression," Wells said. "We see persistent expression of genes that are normally active only in differentiated cells, because donor-cell genes are not switched off, and there is also a failure to re-activate embryonically active genes.

"The consequences include high rates of embryonic loss in pregnancy, higher birth weight, and a higher rate of perinatal and longer-term mortality."

In cattle, 40 to 80 per cent of calves born survived to adulthood. Most showed normal growth, physiology, behaviour, reproduction and productivity. But various clone-associated phenotypes included reduced longevity, reduced immune competency, obesity, and variability between clones from the same animal.

The differences depended on the species, the genotype, the cell type used in SCNT, and the nuclear-transfer and in vitro protocols used. Significantly, the problems of 'cloning syndrome' did not appear to be heritable, confirming they involved epigenetic aberrations, not mutations; these errors were repaired during gametogenesis in affected clones.

"This provides encouragement in all cloning applications, but we need more molecular evidence to determine if there are any longer-term, transgenerational effects," Wells said.

"We need to improve reprogramming of the donor cell, because some effects are due to the particular phase of the donor cycle, and whether the cell is transgenic or not."

For example, experiments with seven different lines of G0 versus G1 phase non-transgenic cells, showed that a significantly higher number of embryos derived from G0 cells developed to term, but the percentage that survived to weaning was then approximately equal -- 17 versus 18 per cent.

For embryos derived from transgenic cells, only 15 per cent of developed to term, and only 5 per cent to weaning age; for embryos developed from transgenic G1 phase cells, 30 per cent developed to term, and 29 per cent to weaning age. "So for non-transgenic embryos, G0 phase cells are superior, but for transgenic cells, G1 is superior. This shows an interaction between donor-cell phase and cell type."

The two major applications of cloning were multiplication of genetically superior animals, and production of novel, transgenic animals, Wells said. "You can take a top Holstein-Friesian dairy sire in New Zealand, and generate three cloned bulls with the same elite genetics for breeding."

Cloning offered even greater value to both the sheep and beef industries, where hundreds of cloned copies of elite, progeny-tested rams or beef bulls could be produced for natural matings.

As with the Enderby Island breed, cloning could also be used to resurrect animals that had died accidentally, or in disease outbreaks, or to conserve and multiply rare breeds or genotypes, such as individuals with strong resistance to internal parasites.

Cloning hybrid animals allowed breeders to maintain favourable heterozygous combinations from outstanding animals, without segregation in the F2 generation.

"All somatic cloning has the drawback of the time required to evaluate the phenotype of the animal, and the time required to create cloned copies," Wells said. "But edible produces from clones, and their clones, does not pose any greater health risk than conventional animal products."