Nine months and twenty days of anxious waiting, but it was worthwhile. On July 11 last, in Jaboticabal, at the Veterinary Hospital of the São Paulo State University (Unesp), the first Brazilian clone, the heifer Penta, was born, by Cesarean section. The two previous clones, Vitória, a heifer from Embrapa in Brasilia and Marcolino a calf from the College of Veterinary Medicine and Zootechny of the University of São Paulo (USP), were cloned from the cells of an embryo and a fetus, respectively. These processes are regarded as more simple than the one used by the researchers from Unesp. The cells from fetuses and embryos reprogram themselves more easily than a differentiated cell (obtained from an adult animal). Among other factors, nuclear reprogramming involves complex reactivation and expression of genes able to sustain the development of a new individual.
The feat belonged to the team of veterinarian Joaquim Mansano Garcia, a professor at the Veterinary Medicine and Animal Reproduction Department of the School of Agrarian and Veterinary Sciences (FCAV) at Unesp, in Jaboticabal. The research enjoyed finance from FAPESP, through of a thematic project coordinated by Garcia, which started in 1998 and is forecast to finish in 2003.
According to the professor, cloning is just one of the strands of the project, which has wider objectives. “It aims to understand the formation of Brazilian Nelore cattle, the most numerous in the country, and its main focus will be the study of mitochondria”, explains the researcher. Mitochondria are regarded as the power plant of cells, responsible for the respiration and survival of the cell. Alterations to the functioning of this organelle can jeopardize the metabolism of the cell, and, in some situations, predispose towards degenerative diseases.
According to the researcher from Unesp, the studies will provide information for the genetic improvement of the country’s herd of cattle. It is known that beef and dairy cattle of European origin (Bos taurus taurus) show greater sexual precocity and productive capacity, although a lower resistance to diseases and to the heat of the tropics, compared with cattle of Indian origin (Bos taurus indicus), called zebu, which includes the animals of the Nelore race.
“The thematic project seeks to investigate the correlation between the mitochondrial types, an assessment of the influence of the maternal inheritance of thetaurus or indicus mitochondrial DNA, and the characteristics of beef cattle production and sexual precocity”, explains Walt Yamazaki, who is studying for his doctorate and is part of Professor Garcia’s team. The group is also made up of Simone Méo and Christina Ferreira, studying for their doctorates.In the cloning process, the researchers carry out the fusion of a cell of the animal that is going to be cloned (the donor of the nucleus) with an ovule without a nucleus, and hence without genetic material of another animal. This technique is called nuclear transfer. Next, the reconstituted ovule is induced to develop as if it were the result of the fertilization of an ovule by a spermatozoid. For this to happen, it is artificially activated in the laboratory.
The researchers from Unesp used a somatic donor cell with a nucleus with mitochondrial DNA from a Bos indicus cow, and an ovule with Bos taurus mitochondrial DNA. They want to assess what happens with these Bos indicus mitochondrias, inserted with the nucleus of the receptor ovule. In animal clones, it is known that the mitochondria of the ovule prevail in the case of Penta, almost 3% of its mitochondrial DNA are from the Nelore (Bos indicus) cow. As was it predicted , the mitochondria of the ovule from the European cattle prevailed, although it is a clone of a zebu.
The researchers have now begun to monitor the heifer Penta and its future descendants. “Among other things, we want to know what happens to Penta’s gametes (reproductive cells). Is she going to reproduce just gametes with mitochondria present in the donor cell of the nucleus or of the ovule?”, asks Garcia.
More broadly, the analysis of the mitochondrial DNA of cattle will provide input that will extrapolate the field of genetic improvement. It will also be useful for understanding certain degenerative diseases in humans, such as Alzheimer’s, related to malfunctioning of the mitochondria. “Our research will provide input for us to understand what happens with human beings”, says Garcia.
The birth of Penta reaffirmed Brazil’s leadership in animal cloning research in Latin America. Unesp’s heifer was born weighing 42 kilos, and is the fruit of an effort that started in 1986, with research related to the fecundation of cattle in vitro. The nucleus of Penta’s genetic material was taken from the tail of a Nelore cow of Pure Imported Origin (that is, whose mother and father have not had any blood from European cattle), 17 years old. The ovule, in turn, was taken from a cow of European origin at the abattoir. The surrogate mother, which received the embryo, was a mixed race zebu-Dutch cow. “We opted for taking fragments of the tissue of the skin from the fold in the tail as a matter of convenience”, Garcia said. “It could have been from any other place, like the ear, but it was easier to take the material out with a simple epidural anesthetic (applied in the tail and covering the animal’s hindquarters)”.
Starting off with these cells, the researchers produced 19 embryos in the laboratory. Next, they were implanted in 11 cows, and some of them received more than one embryo – Penta’s mother, for example, received two. Three cows started their gestation, but only one of them carried out the process normally, up to the end. As soon as she was born, Penta was rejected by her surrogate mother”. A zebu cow needs to feel the smell of its offspring and to lick it as soon as it is born, otherwise it will not recognize it and will reject it”, Professor Garcia explained. After being given a feeding bottle for six days, Penta accepted the udders of two cows of the Dutch race that did not reject her.
The Jaboticabal project used the same technique used in cloning Dolly the ewe, which took place in 1996, in Scotland. The researchers from Unesp, however, innovated by using strontium chloride, combined with a drug called ionomycin, to activate the reconstituted ovule. In earlier experiments, ionomycin was used in association with another drug, 6DMAP. “Strontium chloride produces an effect that is closer to normal fecundation by a spermatozoid”, explains Yamazaki, who was directly responsible for Penta’s cloning. “The studies show that strontium shows a better result for the development of embryo clones reconstituted with cells from an adult animal”.
Up to this moment, the scientists from Unesp have already received over R$ 900,000 from FAPESP, which was used in the acquisition of equipment and materials, the payment of grants for researchers, and expenses with imports. Among the important apparatuses, three were essential for the success of the research: a microscope with a system of image processing and micromanipulation, a production system with pipettes, and cell electrofusion apparatus, which makes it possible to introduce nuclear material into the ovule.
The thematic project also enjoys the participation of other institutions: the Genetics and Physiology Departments of the Faculty of Medicine and the Toxicology Department of the School of Pharmaceutical Sciences, both from USP in Ribeirão Preto, and the School of Zootechny and Food Engineering of USP in Pirassununga. All together, they scored a fantastic goal, in such a delicate and sophisticated subject as animal cloning.
Study of the Function and Heritage of Mitochondrial DNA (mtDNA) in Cattle: An Animal Model Produced with Nelore (nº 98/11783-4); Modality Thematic Project; Coordinator Joaquim Mansano Garcia – Unesp Jaboticabal; Investment R$ 440,023.93 and US$ 229,318.57