The effect of the number of transferred embryos, the interval between nuclear transfer and embryo transfer, and the transfer pattern on pig cloning efficiency.

To improve the efficiency of producing cloned pigs, we investigated the influence of the number of transferred embryos, the culturing interval between nuclear transfer (NT) and embryo transfer, and the transfer pattern (single oviduct or double oviduct) on cloning efficiency. The results demonstrated that transfer of either 150-200 or more than 200NT embryos compared to transfer of 100-150 embryos resulted in a significantly higher pregnancy rate (48 ± 16, 50 ± 16 vs. 29 ± 5%, p<0.05) and average litter size (4.1 ± 2.3, 7 ± 3.6 vs. 2.5 ± 0.5). In vitro culture of reconstructed embryos for a longer time (40 h vs. 20 h) resulted in higher (p<0.05) pregnancy rate (44 ± 9 vs. 31 ± 3%) and delivery rate (44 ± 9 vs. 25 ± 9%). Furthermore, double oviductal transfer dramatically increased pregnancy rate (83 ± 6 vs. 27+8%, p<0.05), delivery rate (75 ± 2 vs. 27+8%, p<0.05) and average litter size (6.5 ± 2.8 vs. 2.6 ± 1.2) compared to single oviductal transfer. Our study demonstrated that an improvement in pig cloning efficiency is achieved by adjusting the number and in vitro culture time of reconstructed embryos as well as the embryo transfer pattern.

Longitudinal study of reproductive performance of female cattle produced by somatic cell nuclear transfer.

The objective of this study was to determine whether or not reproductive performance in cattle produced by somatic cell nuclear transfer (SCNT) is significantly different from that of their genetic donors. To address this question, we directed two longitudinal studies using different embryo production procedures: (1) superovulation followed by artificial insemination (AI) and embryo collection and (2) ultrasound-guided ovum pick-up followed by in vitro fertilization (OPU-IVF). Collectively, these two studies represent the largest data set available for any species on the reproductive performance of female clones and their genetic donors as measured by their embryo production outcomes in commercial embryo production program. The large-scale study described herein was conducted over a six-year period of time and provides a unique comparison of 96 clones to the 40 corresponding genetic donors. To our knowledge, this is the first longitudinal study on the reproductive performance of cattle clones using OPU-IVF. With nearly 2,000 reproductive procedures performed and more than 9,200 transferable embryos produced, our observations show that the reproductive performance of cattle produced by SCNT is not different compared to their genetic donors for the production of transferable embryos after either AI followed by embryo collection (P = 0.77) or OPU-IVF (P = 0.97). These data are in agreement with previous reports showing that the reproductive capabilities of cloned cattle are equal to that of conventionally produced cattle. In conclusion, results of this longitudinal study once again demonstrate that cloning technology, in combination with superovulation, AI and embryo collection or OPU-IVF, provides a valuable tool for faster dissemination of superior maternal genetics.

Fertilization rates and in vitro embryo production using sexed or non-sexed semen selected with a silane-coated silica colloid or Percoll.

The aim of this study was to evaluate sperm fertilization rates and in vitro embryo development rates for sexed and non-sexed semen selected using a silane-coated silica colloid method (Isolate) or Percoll. Frozen/thawed, sexed and unsexed semen samples from four Holstein bulls were randomly allocated to one of two different density gradient selection methods. Sperm quality (motility, concentration, morphology and membrane integrity) were evaluated and compared before and after sperm selection. Sperm motility and morphology improved (P < 0.005) after the sperm selection process with no differences between the two methods. For non-sexed semen, Percoll gradient increased the mean (± SEM) percentage of sperm recovered (57.3 ± 2.8) compared to Isolate (46.0 ± 1.8; P < 0.01). However, membrane integrity was higher after Isolate than Percoll (sexed semen: 41.0 ± 0.6 vs. 38.8 ± 0.8 and non-sexed semen 60.8 ± 1.6 vs. 58.8 ± 0.5; P < 0.05). The percentage of blastocysts produced was higher when either sexed or non-sexed semen was selected by Isolate (14.0 ± 1.0; 22.0 ± 1.1) than by Percoll (10.5 ± 1.5; 17.0 ± 2.1, respectively; P < 0.05). In summary, Isolate was a more effective method for the recovery of high quality sperm for in vitro fertilization embryo production.

Application of genetically modified and cloned pigs in translational research.

Pigs are increasingly being recognized as good large-animal models for translational research, linking basic science to clinical applications in order to establish novel therapeutics. This article reviews the current status and future prospects of genetically modified and cloned pigs in translational studies. It also highlights pigs specially designed as disease models, for xenotransplantation or to carry cell marker genes. Finally, use of porcine somatic stem and progenitor cells in preclinical studies of cell transplantation therapy is also discussed.

Future and applications of cloning.

The birth of viable offspring from somatic cell nuclear transfer (SCNT) in mammals caused a major re-examination of the understanding of the commitment of cells to specific tissue lineages during differentiation. The questions of whether cells undergo dedifferentiation or transdifferentiation during the development of offspring and how these changes are controlled is a source of ongoing debate that is yet to be resolved. Irrespective of the outcome of this debate, it is clear that cloning using SCNT has a place and purpose in the future of research and animal breeding. The future uses of SCNT could include the production of transgenic mice, the production of transgenic livestock and assisting with the re-establishment of endangered species. Human medicine also would benefit from future use of SCNT because it would allow the production of patient-specific embryonic stem cells.

Using therapeutic cloning to fight human disease: a conundrum or reality?

The development and transplantation of autologous cells derived from nuclear transfer embryonic stem cell (NT-ESC) lines to treat patients suffering from disease has been termed therapeutic cloning. Human NT is still a developing field, with further research required to improve somatic cell NT and human embryonic stem cell differentiation to deliver safe and effective cell replacement therapies. Furthermore, the implications of transferring mitochondrial heteroplasmic cells, which may harbor aberrant epigenetic gene expression profiles, are of concern. The production of human NT-ESC lines also remains plagued by ethical dilemmas, societal concerns, and controversies. Recently, a number of alternate therapeutic strategies have been proposed to circumvent the moral implications surrounding human nuclear transfer. It will be critical to overcome these biological, legislative, and moral restraints to maximize the potential of this therapeutic strategy and to alleviate human disease.

The egg-sharing model for human therapeutic cloning research: managing donor selection criteria, the proportion of shared oocytes allocated to research, and amount of financial subsidy given to the donor.

Recent advances in human therapeutic cloning made by Hwang and colleagues have opened up new avenues of therapy for various human diseases. However, the major bottleneck of this new technology is the severe shortage of human donor oocytes. Egg-sharing in return for subsidized fertility treatment has been suggested as an ethically justifiable and practical solution to overcome the shortage of donor oocytes for therapeutic cloning. Because the utilization of shared oocytes in therapeutic cloning research does not result in any therapeutic benefit to a second party, this would necessitate a different management strategy compared to their use for the assisted conception of infertile women who are unable to produce any oocytes of their own. It is proposed that the pool of prospective egg-sharers in therapeutic cloning research be limited only to younger women (below 30 years of age) with indications for either male partner sub-fertility or tubal blockage. With regards to the proportion of the shared gametes being allocated to research, a threshold number of retrieved oocytes should be set that if not exceeded, would result in the patient being automatically removed from the egg-sharing scheme. Any excess supernumerary oocyte above this threshold number can be contributed to science, and allocation should be done in a randomized manner. Perhaps, a total of 10 retrieved oocytes from the patient may be considered a suitable threshold, since the chances of conception are unlikely to be impaired. With regards to the amount of subsidy being given to the patient, it is suggested that the proportion of financial subsidy should be equal to the proportion of the patient's oocytes being allocated to research. No doubt, the promise of future therapeutic benefit may be offered to the patient instead of financial subsidy. However, this is ethically controversial because therapeutic cloning has not yet been demonstrated to be a viable model of clinical therapy and any promises made to the patient might turn out to be illusionary. Hence, it is proposed that a tangible financial subsidy on the medical fees might be the better option for the patient's welfare.

The contribution of farm animals to human health.

Farm animals and their products have a longstanding and successful history of providing significant contributions to human nutrition, clothing, facilitation of labour, research, development and medicine and have thus been essential in improving life expectancy and human health. With the advent of transgenic technologies the potential of farm animals for improving human health is growing and many areas remain to be explored. Recent breakthroughs in reproductive technologies, such as somatic cloning and in vitro embryo production, and their merger with molecular genetic tools, will further advance progress in this field. Here, we have summarized the contribution of farm animals to human health, covering the production of antimicrobial peptides, dietary supplements or functional foods, animals used as disease models and the contribution of animals to solving urgent environmental problems and challenges in medicine such as the shortage of human cells, tissues and organs and therapeutic proteins. Some of these areas have already reached the level of preclinical testing or commercial application, others will be further advanced only when the genomes of the animals concerned have been sequenced and annotated. Provided the necessary precautions are being taken, the transmission of pathogens from animals to humans can be avoided to provide adequate security. Overall, the promising perspectives of farm animals and their products warrant further research and development in this field.

Obesity--a chronic health problem in cloned mice?

The majority of cloned animals derived by nuclear transfer die during gestation and display neonatal abnormalities. However, because of the long generation interval of cloned animal species, the long-term consequences of cloning on health have been difficult to access. Recent studies in mice have provided evidence that cloned animals might have chronic health problems such as obesity. Obesity in cloned mice is likely to reflect epigenetic abnormalities that arise partly from inadequate nuclear programming; these obese mice display a unique phenotype that is suggestive of a defect other than malfunction of the leptin-melanocortin system, which occurs in most rodent models of obesity and in human obesity.

Infectious disease issues in xenotransplantation.

Xenotransplantation, the transplantation of living organs, tissues, or cells from one species to another, is viewed as a potential solution to the existing shortage of human organs for transplantation. While whole-organ xenotransplantation is still in the preclinical stage, cellular xenotransplantation and extracorporeal perfusion applications are showing promise in early clinical trials. Advances in immunosuppressive therapy, gene engineering, and cloning of animals bring a broader array of xenotransplantation protocols closer to clinical trials. Despite several potential advantages over allotransplantation, xenotransplantation encompasses a number of problems. Immunologic rejection remains the primary hindrance. The potential to introduce infections across species barriers, another major concern, is the main focus of this review. Nonhuman primates are unlikely to be a main source for xenotransplantation products despite their phylogenetic proximity to humans. Genetically engineered pigs, bred under special conditions, are currently envisaged as the major source. Thus far, there has been no evidence for human infections caused by pig xenotransplantation products. However, the existence of xenotropic endogenous retroviruses and the clinical evidence of long-lasting porcine cell microchimerism indicate the potential for xenogeneic infections. Thus, further trials should continue under regulatory oversight, with close clinical and laboratory monitoring for potential xenogeneic infections.