Biomedical subjects

R Duchi

Publications and source records attributed to R Duchi.

6 recordsLinked to original sources

PubMed2006

Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer.

Development of assisted reproductive technologies in horses has been relatively slow compared to other domestic species, namely ruminants and pigs. The scarce availability of abattoir ovaries and the lack of interest from horse breeders and breed associations have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established followed by the application of ICSI to achieve fertilization in vitro. Embryo culture was initially performed in vivo, in the mare oviduct or in the surrogate sheep oviduct, to achieve the highest embryo development, in the range of 18-36% of the fertilised oocytes. Subsequently, the parallel improvement of in vitro oocyte maturation conditions and embryo culture media has permitted high rates of embryo development from in vitro matured and in vitro cultured ICSI embryos, ranging from 5 to 10% in the early studies to up to 38% in the latest ones. From 2003, with the birth of the first cloned equids, the technology of somatic cell nuclear transfer has also become established due to improvement of the basic steps of embryo production in vitro, including cryopreservation. Pregnancy and foaling rates are still estimated based on a small number of in vitro produced equine embryos transferred to recipients. The largest set of data on non-surgical embryo transfer of in vitro produced embryos, from ICSI of both abattoir and in vitro-matured Ovum Pick Up (OPU) oocytes, and from somatic cell nuclear transfer, has been obtained in our laboratory. The data demonstrate that equine embryos produced by OPU and then cryopreserved can achieve up to 69% pregnancy rate with a foaling rate of 83%. These percentages are reduced to 11 and 23%, respectively, for cloned embryos. In conclusion, extensive evidence exists that in vitro matured equine oocytes can efficiently develop into viable embryos and offspring.

Animals↗

PubMed2005

Establishment, differentiation, electroporation, viral transduction, and nuclear transfer of bovine and porcine mesenchymal stem cells.

Mesenchymal stem cells (MSCs) reside in the bone marrow and have the potential for multilineage differentiation, into bone, cartilage, and fat, for example. In this study, bovine and porcine MSCs were isolated, cultured to determine their replication ability, and differentiated with osteogenic medium and 5-azacytine. Both bovine and porcine undifferentiated MSCs were electroporated and virally transduced to test the efficiency of genetic modification and the maintainance of differentiation ability thereafter. Nuclear transfer experiments were carried out with bovine and porcine MSCs, both at the undifferentiated state and following differentiation. Our results indicate that bovine and porcine MSCs have limited lifespans in vitro--approximately 50 population doublings. They can be efficiently differentiated and characterized along the osteogenic lineage by morphology, alkaline phosphatase, Von Kossa, oil red stainings, and RT-PCR. Electroporation and selection induce high levels of EGFP expression in porcine but not in bovine MSCs. Following genetic modification, MSCs retain their pluridifferentiation ability as parental cells. Cloned embryos derived from bovine and porcine undifferentiated MSCs and their derivatives along the osteogenic lineage give rise to consistently high preimplantation development comparable to adult fibroblasts.

Animals↗

PubMed2004

Production and quality of bovine oocytes and embryos.

Many factors influence the efficiency of the in vitro embryo production technology in cattle but the most important are the physiological conditions of the donor and the culture protocols for oocyte maturation and fertilization and for embryo culture from zygote to blastocyst. Therefore, general factors such as age, body conditions and herd management play a pivotal role together with more specific factors such as reproductive soundness and ovarian cyclicity. Given that good quality and competent oocytes are available a complex series of processes, including oocyte maturation, fertilization and culture of the derived zygotes, must be completed to generate viable embryos.

Animals↗

PubMed2003

Bovine embryo technologies.

Embryo technologies are a combination of assisted reproduction, cellular and molecular biology and genomic techniques. Their classical use in animal breeding has been to increase the number of superior genotypes but with advancement in biotechnology and genomics they have become a tool for transgenesis and genotyping. Multiple ovulation and embryo transfer (MOET) has been well established for many years and still accounts for the majority of the embryos produced worldwide. However, no progress has been made in the last 20 years to increase the number of transferable embryos and to reduce the side effects on the reproductive performance of the donors. In vitro embryo production (IVP) is a newer and more flexible approach, although it is technically more demanding and requires specific laboratory expertise and equipment that are most important for the quality of the embryos produced. Somatic cell cloning is a rapidly developing area and a very valuable technique to copy superior genotypes and to produce or copy transgenic animals. More knowledge in oocyte and embryo biology is expected to shed new light on the early developmental events, including epigenetic changes and their long lasting effect on the newborn.Embryo technologies are here to stay and their use will increase as advances in the understanding of the mechanisms governing basic biological processes are made.

Animals↗

PubMed2001

Embryo production by ovum pick up from live donors.

Embryo production by in vitro techniques has increased steadily over the years. For cattle where this technology is more advanced and is applied more, the number of in vitro produced embryos transferred to final recipients was over 30,000 in 1998. An increasing proportion of in vitro produced embryos are coming from oocytes collected from live donors by ultrasound-guided follicular aspiration (ovum pick up, OPU). This procedure allows the repeated production of embryos from live donors of particular value and is a serious alternative to superovulation. Ovum pick up is a very flexible technique. It can be performed twice a week for many weeks without side effects on the donor's reproductive career. The donor can be in almost any physiological status and still be suitable for oocyte recovery. A scanner with a sectorial or convex probe and a vacuum pump are required. Collection is performed with minimal stress to the donor. An average of 8 to 10 oocytes are collected per OPU with an average production of 2 transferable embryos. The laboratory production of embryos from such oocytes does not differ from that of oocytes harvested at slaughter as the results after transfer to final recipients. For other species such as buffalo and horses OPU has been attempted similarly to cattle and data will be presented and reviewed. For small ruminants, laparotomy or laparoscopy seems the only reliable route so far to collect oocytes from live donors.

Animals↗

PubMed1999

Mammalian leukocytes contain all the genetic information necessary for the development of a new individual.

We have used leukocytes and oocytes from commercially slaughtered animals to clone a progeny tested Brown Swiss bull. Mononuclear cells were separated from the heparinized blood of the donor male on a Histopaque gradient and cryopreserved. The nuclei of thawed leukocytes were directly microinjected into enucleated Holstein Friesian oocytes that were subsequently activated. Development to morula was 23% and to blastocysts was 17%. Some of the cloned compacting morulae were subjected to a second round of nucleus transfer by fusion of individual blastomeres to enucleated oocytes. Development of these second generation embryos to the blastocyst stage was 19%. Following embryo transfer of 50 blastocysts to 50 recipient heifers (31 from first generation and 19 from second generation), 28 pregnancies were established as evidenced by fetal heartbeat at 35 days. A high proportion of the pregnancies established were lost by day 45. One fetus from a second generation embryo developed to term. The phenotype (Brown Swiss) and DNA analysis (11 microsatellites on 11 different chromosomes) of the resultant normal healthy calf confirmed its identity to the donor sire. The ability to clone animals from hematopoietic cells that can be easily collected and cryopreserved from any donor irrespective of species, age, or sex has important implications for the preservation of genetic resources from a wide variety of animals in the animal breeding and artificial insemination industries and for human medicine.

Animals↗