Cloned milk and cereals Cells taken from ovaries in a Delhi abattoir result in India's - and the world's - first cloned water buffalo in a modest laboratory in Karnal. Anand Sankar comes away charmed by Garima. Garima’s might be an enviable life. Her sanitised environment is air-conditioned, she’s fussed over and clearly enjoys it, she rushes to meet her visitors, and her diet is carefully supervised. Yet, two months after her birth, she has basked in rather a muted limelight, as indeed has the modest laboratory where this water buffalo was cloned. It is more than a decade since Dolly the sheep, the world’s first large cloned mammal, made headlines. Garima’s arrival on June 6, 2009, has created rather less fuss, not just because cloning has grown in the intervening decade, with even monkeys, mules, horses and camels recently added to the list in scientific laboratories around the world. That is a long step from the time that a tadpole, in 1952, and a carp, in 1963, became the first cloned living beings in the history of mankind. Since then, controlled experiments have been used to collect data and research species, with large-scale cloning left to commercial interests. For now, that is restricted to pet cats and dogs in California, at prices starting at $20,000. Garima cost only a fraction of such sums. In fact, the very existence of Garima, India’s first surviving cloned living being, is a marvellous result not of the technology that made it possible — which is growing by leaps and bounds — as much as it is of the modest scale and spend of the operation. Garima was created by scientists at the National Dairy Research Institute (NDRI), located in Karnal, a nondescript town in Haryana — hardly the place you’d associate with cutting-edge scientific research. “This is only the first step in our project,” beams Dr Suresh K Singla, the lead scientist, betraying a hint of relief. “We are hoping to study the calf for its whole life.” Garima will have to cross several hurdles for the experiment to prove a success. For example, an earlier cloned calf, born in February, died within 24 hours of its birth. Dolly’s story was path-breaking because it saw textbook theory being converted to life. But Garima’s story is no less ingenious, if typically Indian. The water buffalo calf began not, as many might presume, in a spotless laboratory, but in a rather more unhygienic abattoir on the outskirts of New Delhi. The egg which produced Garima (Step Zero in lab parlance) was taken from buffalos slaughtered for their meat. The ovaries of the buffalos — usually discarded as entrails — were harvested by the scientists and transported to the research institute. This has been the routine for the cloning project, which began way back in 1994. The eggs, when cleaned and separated, made their way into a tiny lab in the heart of the NDRI campus. Here the team of scientists — Drs Suresh K Singla, R S Manik, M S Chauhan, P Palta, Shiv Prasad, R A Shah and Aman George — went to work with a technique they had adapted especially for Indian conditions. “Initially, we tried the Dolly method. But after several years of frustrating failures, the method was abandoned,” recalls Dr Singla. Their technique has been christened the hand-guided method. The main drawback of the Dolly method was the requirement of highly specialised, thus prohibitively expensive, laboratory tools, to which the Indian team realised it was unlikely to have access. It instead decided to adapt the efforts of Gabor Vajta, professor and senior scientist at the Danish Institute of Agricultural Sciences (DIAS) who had sought to lower the cost of cloning. Vajta demonstrated his method with horses, cattle and pigs. Hand-guided cloning needs just a micro manipulator, a device used to work on microscopic organisms, and that was available at NDRI. New age baby boom A step-by-step guide to the hand-guided method used in the Karnal laboratory Step 1: Two eggs are taken and the “egg shells” are removed using enzymes Step 2: The nucleus material “yolk” of the eggs is removed. To do that, enzymes are used to push it to one side. The resulting “bulge” is cut with a finely sharpened micro blade Step 3: The nucleus of the chosen cell (taken from the animal to be cloned) is extracted. Skin cells are chosen, because they are the least specialised Step 4: The nucleus is sandwiched between the two eggs and a low-voltage electric current is applied. This results in all the material fusing Step 5: The resulting egg with the foreign nucleus is incubated. If it becomes a healthy, growing embryo, it is chosen. The embryo, before it becomes 100 cells in size, is further manually divided to form multiple identical embryos (identical twins) Step 6: Four identical embryos are implanted in the uterus of each buffalo chosen as a surrogate mother. The buffalos have had their fertility monitored and are treated to ensure they don’t reject the embryos. Step 7: If a pregnancy results in the implanted buffalos, then you have a good chance of a cloned calf being born The Dolly method involves taking an egg, sucking out its yolk and replacing it with the DNA of the animal to be cloned. The resulting egg is zapped with electricity to kick-start its growth into an embryo. The scientists at Karnal say this method, even with top-notch equipment, has a high rate of failure. By contrast, the hand-guided method has a higher success rate in large mammals. The buffalo is the fourth species to be cloned using the hand-guided method, but it certainly isn’t simple. One peculiar problem scientists face is when the enzymes they are using in the process do not work as they should. NDRI troubleshooters traced the cause to the toxicity in the imported plastic lab apparatus, which was reacting with the enzymes. (Lab apparatus is supposd to be inert.) As a result, all lab apparatus had to be tested for toxicity before use. Not all was fine even once pregnancy was confirmed and a calf was about to be born. The first cloned calf, the scientists say, died as a result of pneumonia, which was traced to it having ingested fluids into the lungs during the birthing process. Garima is from the popular Murrah breed of buffalos. Scientists say they have recorded yields of up to 2,000 litres of milk in a single standard lactation period of 310 days. In India, 55 per cent of total milk production is from buffalos, and buffalo milk has the highest SNF (solids not fat) count and fatty content. According to agriculture ministry statistics, out of around 100 million livestock in the country, 47 million are buffalos, 11 million are cross-breeds and 45 million belong to indigenous cattle varieties. On a global scale, India has about 16 per cent of the world’s cattle population, 56 per cent of its buffalo wealth and 16.2 per cent of the goat population. “The ultimate aim is to multiply the best bulls and females. You can say it is a primitive kind of Xerox machine. It will ensure faster propagation of chosen traits. Sex selection is also easily possible as the calf is in every respect an identical copy of the original,” says Dr Singla. In February, when the first buffalo was born, the project was granted Rs 7 crore as extra funding under the National Agriculture Innovation Project (NAIP). “The sum was applied for when the gestation period of the calf began, and it took just as long to materialise,” NDRI scientists say with a laugh. The sum is considered a record of sorts for a single agricultural research project in India. The next step now is to study Garima to see whether she develops any abnormalities later in life. Dolly the sheep did not live her full lifespan: her cells aged prematurely because her clone parent was an adult sheep. In the case of Garima, the scientists say they might have circumvented the problem because they took the parent cells from an unborn foetus. Thus, a full lifespan can be expected. India currently has no regulations for animal cloning. The Department of Biotechnology only regulates human stem cell research, and transgenic research — where traits of different species are mixed. An example of a transgenic cross would be rice with scorpion genes, or a buffalo engineered to produce some medicine in its milk. NDRI scientists say they are involved only in “pure cloning” for which they have been given the nod by the agriculture ministry. But they admit that transgenic research is the future, though they will not start transgenic research without approval from the Department of Biotechnology. How long is it before we find milk from cloned buffalos on the breakfast table? In the US, the Food and Drug Administration in January 2008 ruled that “meat and milk from cow, pig, and goat clones and the offspring of any animal clones are as safe as food we eat every day”. The ruling allowed products from cloned animals to be sold in retail without any special labelling. But it is still too early for such products to have made it to supermarket shelves. In India, scientists say cloned buffalo milk is still years away. “Commercialisation is not our priority,” says Dr Singla. “It is first important to establish a success rate.” Can cloning save the tiger? Cloning experiments — and success — have led to hopes that endangered, even extinct, species, can be revived. For now, companies in the USA are working on isolating the best breeds of cattle and poultry to clone so that farmers can raise the quality of their livestock. But scientists at the National Dairy Research Institute say that each species of animal has different characteristics, and one cloning method cannot be said to suit them all. But can it come to the rescue of, say, the royal Bengal tiger? The hand-guided method of cloning, scientists warn, has only been proven with large mammals whose fertility characteristics are known. There is also the problem of harvesting eggs. It is easy enough to get a large number of ovaries from buffalos since they are slaughtered for meat, but extracting eggs from live animals produces only a few eggs at a time. Any tiger-cloning project would entail enormous logistics to locate sufficient donor eggs and parent cells. Tiger conservationists add another dimension. They say the gene pool of wild tigers is already tiny, and cloning them would not diversify the gene pool. There is also an element of the unknown if captive bred tigers are released into the wild. Conservationists say the best hope is to preserve the tiger’s habitat and hope the population recovers naturally.