Roberto Duchi

Cellular and Molecular Deviations in Bovine In Vitro-Produced Embryos Are Related to the Large Offspring Syndrome

Abstract The large offspring syndrome (LOS) is observed in bovine and ovine offspring following transfer of in vitro-produced (IVP) or cloned embryos and is characterized by a multitude of pathologic changes, of which extended gestation length and increased birthweight are predominant features. In the present study, we used bovine blastocysts to analyze cellular parameters, i.e., the number of cells in Day 7 blastocysts and the size of Day 12 elongating blastocysts, and molecular parameters, i.e., the relative abundance of developmentally important genes: glucose transporter (Glut) 1, Glut-2, Glut-3, Glut-4, heat shock protein (Hsp) 70.1, Cu/Zn-superoxide dismutase (SOD), histone H4.1, basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF) I receptor (R), and IGFII-R. Some blastocysts were produced by in vitro maturation and fertilization followed by in vitro culture in synthetic oviduct fluid medium supplemented with BSA or human serum or by in vivo culture in the sheep oviduct. Other blastocysts were derived in vivo from the uterine horns of superovulated donors. The findings made in the early embryos were related to a representative number of calves obtained from each production system and from artificial insemination (AI). In vitro culture of bovine embryos in the presence of high concentrations of serum or BSA significantly increased the number of cells in Day 7 blastocysts, the size of blastocysts on Day 12, and the relative abundance of the transcripts for Hsp70.1, Cu/Zn-SOD, Glut-3, Glut-4, bFGF, and IGFI-R when compared with embryos from the in vivo production groups. Birthweights of calves derived from IVP embryos were significantly higher than those of calves derived from sheep oviduct culture, superovulation, or AI. The results support the hypothesis that persistence of early deviations in development is causally involved in the incidence of LOS, in particular in increased birthweights. The cellular and molecular parameters analyzed in this study can be considered early markers of LOS in cattle.

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 donors 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.

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.

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.

Developmental Potential of Bovine Androgenetic and Parthenogenetic Embryos: A Comparative Study

Abstract In this study, we compared the developmental capacity of bovine haploid and diploid androgenetic and parthenogenetic embryos obtained by different methods. Androgenetic embryos were produced by piezo-intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF) of enucleated oocytes with or without subsequent pronuclear transfer from one haploid zygote to another. Parthenogenetic embryos were obtained by activation of matured oocytes by ionomycin combined with cycloheximide or 6-dimethylaminopurine (DMAP) treatment. Only few cleaved androgenetic haploid embryos were able to compact (2.7%) and to form blastocysts (1.8%), while significantly more haploid parthenogenotes underwent compaction (24–37%) and a minority developed to blastocysts at different rates, depending on the activation procedure (cycloheximide 3%, 6-DMAP 14.5%). By contrast, development to blastocyst of diploid androgenotes, cloned androgenetic embryos, and parthenogenotes (31%, 39%, and 43%, respectively) was similar to IVF control embryos (35%). Cell number on Day 7 was higher for IVF blastocysts and decreased in consecutive order in diploid androgenotes, diploid parthenogenotes, and haploid uniparental embryos. Following transfer of diploid androgenetic embryos, a pregnancy was established and maintained up to Day 28.

Transgene Expression of Green Fluorescent Protein and Germ Line Transmission in Cloned Pigs Derived from In Vitro Transfected Adult Fibroblasts

The pig represents the xenogeneic donor of choice for future organ transplantation in humans for anatomical and physiological reasons. However, to bypass several immunological barriers, strong and stable human genes expression must occur in the pigs organs. In this study we created transgenic pigs using in vitro transfection of cultured cells combined with somatic cell nuclear transfer (SCNT) to evaluate the ubiquitous transgene expression driven by pCAGGS vector in presence of different selectors. pCAGGS confirmed to be a very effective vector for ubiquitous transgene expression, irrespective of the selector that was used. Green fluorescent protein (GFP) expression observed in transfected fibroblasts was also maintained after nuclear transfer, through pre- and postimplantation development, at birth and during adulthood. Germ line transmission without silencing of the transgene was demonstrated. The ubiquitous expression of GFP was clearly confirmed in several tissues including endothelial cells, thus making it a suitable vector for the expression of multiple genes relevant to xenotransplantation where tissue specificity is not required. Finally cotransfection of green and red fluorescence protein transgenes was performed in fibroblasts and after nuclear transfer blastocysts expressing both fluorescent proteins were obtained.

Comparison of microinjection (piezo-electric) and cell fusion for nuclear transfer success with different cell types in cattle.

Amongst the many variables that can determine success of cloning, the source of nuclei, the procedure used for nuclear transfer, and the activation of the reconstructed embryo are very important aspects. In this study, we have compared the two most common procedures for transferring nuclei to enucleated oocytes--cell fusion (CF) and piezoelectric microinjection (PEM) using different somatic cells--and we have investigated the effect of different activation procedures. Granulosa cells and fibroblasts were grown to confluency or in low serum to induce a quiescent state, while lymphocytes were thawed immediately prior to use. Enucleated oocytes were reconstructed either with CF or PME by 21-23 h postmaturation. For cell fusion, one pulse of 1 kVolt/cm for 30 microsec was used; for PEM, the cell membrane was broken by repeated pipetting and transferred in a 12% PVP solution to facilitate injection. Manipulated oocytes were activated with ionomycin and cycloheximide (CHX) or 6-DMAP (DMAP) and cultured in microdrops of SOF-BSA-AA. On day 7 (day 0: nuclear transfer), embryo development was evaluated and embryos were either transferred fresh or were frozen. More embryos were successfully reconstructed with PEM than CF, but a higher number of reconstructed embryos by CF developed to blastocyst at D + 7. In addition, in both systems more embryos were obtained after activation with DMAP than with CHX. The transfer of 141 embryos to recipients resulted in a pregnancy rate of 50%, and no differences were observed between the source of donor cell, the reconstruction methods, or the activation protocol. Six calves were delivered at term, and four survived. High pregnancy losses were observed throughout the gestation period.

Short-term and long-term effects of embryo culture in the surrogate sheep oviduct versus in vitro culture for different domestic species.

The culture of early embryos in the surrogate xeno-oviduct was first developed in the early 1950s to allow transport of embryos at long distances. Later, it was applied to the study of culture requirements of the early embryo especially that of bovine origin. In this article, we review the data available on the culture of in vitro-matured and in vitro-fertilized embryos of Bos taurus, Sus scrofa, Equus caballus and Ovis aries in the surrogate sheep oviduct compared with data on in vitro culture in different media. Short-term and long-term cellular and molecular effects are described mainly for the bovine species where more extensive use of this technique has been made. A comparison with in vitro culture in various conditions and species indicate that embryos cultured in the sheep oviduct have close similarities to totally in vivo-derived embryos. The data provided demonstrate that the technique of in vivo culture in the surrogate sheep oviduct is versatile and allows a high rate of embryonic development in all species examined.

Somatic cell nuclear transfer and transgenesis in large animals: current and future insights

Somatic cell nuclear transfer (SCNT) was first developed in livestock for the purpose of accelerating the widespread use of superior genotypes. Although many problems still exist now after fifteen years of research owing to the limited understanding of genome reprogramming, SCNT has provided a powerful tool to make copies of selected individuals in different species, to study genome pluripotency and differentiation, opening new avenues of research in regenerative medicine and representing the main route for making transgenic livestock. Besides well-established methods to deliver transgenes, recent development in enzymatic engineering to edit the genome provides more precise and reproducible tools to target-specific genomic loci especially for producing knockout animals. The interest in generating transgenic livestock lies in the agricultural and biomedical areas and it is, in most cases, at the stage of research and development, with few exceptions that are making the way into practical applications.

Embryonic genotype and inbreeding affect preimplantation development in cattle

Infertility in cattle herds is a growing problem with multifactorial causes. Embryonic genotype and level of inbreeding are among the many factors that can play a role on reproductive efficiency. To investigate this issue, we produced purebred and crossbred bovine embryos by in vitro techniques from Holstein oocytes and Holstein or Brown Swiss semen and analyzed several cellular and molecular features. In the first experiment, purebred and crossbred embryos, obtained from abattoir oocytes, were analyzed for cleavage, development to morula/blastocyst stages, amino acid metabolism and gene expression of developmentally important genes. The results indicated significant differences in the percentage of compacted morulae, in the expression of three genes at the blastocyst stage (MNSOD, GP130 and FGF4) and in the utilization of serine, asparagine, methionine and tryptophan in day 6 embryos. In the second experiment, bovine oocytes were collected by ovum pick up from ten Holstein donors and fertilized with the semen of the respective Holstein sires or with Brown Swiss semen. The derived embryos were grown in vitro up to day 7, and were then transferred to synchronized recipients and recovered on day 12. We found that purebred/inbred embryos had lower blastocyst rate on days 7-8, were smaller on day 12 and had lower expression of the trophoblast gene PLAC8. Overall, these results indicate reduced and delayed development of purebred embryos compared with crossbred embryos. In conclusion, this study provides evidence that embryo genotype and high inbreeding can affect amino acid metabolism, gene expression, preimplantation development and therefore fertility in cattle.