Paola Turini

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.

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.

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.

Prolonged In Vitro Maturation Time and Increased Intensity of Piezo Pulses During ICSI Enhance Cleavage and Embryo Development in the Horse

The use of assisted reproduction techniques in horses is increasing, in particular in vitro embryo production by OPU and ICSI. Compared to cattle the percentage of oocytes that become transferable blastocysts is lower both under experimental [1] and clinical settings [2]. The horse has peculiar features, showing a varied population of immature oocytes that require different timing for reaching both nuclear and cytoplasmic maturation. Immature oocytes can also be put on hold at room temperature with no loss of viability [3]. Current in vitro maturation protocols entail a 24 to 28 h maturation time assessed by the appearance of the first polar body, despite the fact that the time from hCG administration (considered the trigger for the resumption of meiosis) to ovulation in vivo is 36 to 40 h. Further observations in the clinical setting in our laboratory indicated that some Met II oocytes matured for 24 to 28 h do not undergo cleavage after ICSI, indicating a possible uncoupling between cytoplasmic and nuclear maturation so that the cytoplasm might not yet be ready for activation. In this work, using experimental oocytes from the slaughterhouse in a controlled setting, we tested the hypothesis that increasing the maturation time and adding a mechanical stimulus, being more aggressive during ICSI, could increase both cleavage and embryo development.

203 SINGLE-STEP GENE EDITING OF 3 XENOANTIGENS IN PORCINE FIBROBLASTS USING PROGRAMMABLE NUCLEASES

Programmable nucleases (ZFN, Tal Effector Nucleases, and CRISPR) opened a new era for mammal genome editing, in particular for the pigs used for xenotransplantation. Multiple gene editing events are required both for knockout (KO) of xenoantigens and for targeted integration of human protective genes (Perota et al. 2016 J. Genet. Genomics 43, 233-23). The objective of the present work was to edit selected pig lines to KO the enzymes coding for the most relevant xenoantigens (i.e. GGTA1, CMAH, and B4GalNT2), combining Talens and CRISPR/Cas9 technologies to magnetic beads selection (Li et al. 2013 Xenotransplantation 22, 20-31). Primary porcine adult fibroblasts were transfected using Nucleofector (V-024 program). In a single reaction 2×106 fibroblasts were co-transfected using 2 different sets of TALENS (4μg/set) specific for CMAH (Conchon et al., 2013) and GGTA1 (Perota et al., 2015) genes together with B4GalNT2-specific CRISPR/Cas9 expression vector (2μg; pX330-B4GalNT2; Estrada et al., 2015). Eight days post-transfection (DPT), Gal-/- cells were selected initially using biotin-conjugated IB4 lectin (Sigma, St. Louis, MO, USA) and magnetic beads (Dynabeads M-280, Thermo Fisher Scientific, Waltham, MA, USA). The selected cells were then plated on 150-mm Petri dishes (200 cells/dish) and cultured for 10 days. Selected colonies were expanded for PCR analysis and cryopreserved for somatic cell nuclear transfer (SCNT). All colonies were analysed by PCR for CMAH gene and their resulting products were digested with HindIII (HindIII-RFLP). Colonies that lost wild-type HindIII as a consequence of Talens effected deletion were PCR characterised for GGTA1, selecting those that had detectable Indels after gel electrophoresis and finally analysed by PCR for B4GalNT2. All PCR products were validated by sequencing for all the 3 genes of interest (TopoTA, Thermo Fisher Scientific). Selected colonies were used as nuclear donors for SCNT (Lagutina et al., 2006). Eight DPT we obtained 3.45×106 cells. About 6.0×103 Gal-negative cells (0.17%) were collected from the supernatant after magnetic beads separation. Eighteen DPT, 120 colonies were picked up and their HindIII-RFLP analyses on CMAH gene revealed that 22 colonies (18.3%) were KO for both CMAH alleles. Of these 22 colonies following electrophoretic analyses of GGTA1-PCR products, 13 colonies had detectable Indels. These 13 colonies were finally PCR analysed and sequenced for B4GalNT2 and sequenced. Final sequencing results confirmed that 2 colonies (1.6%) resulted in KO for the 3 genes. Three different zona-free SCNT experiments were done and 579 reconstructed embryos were obtained. On Day 7, 322 morulae or blastocysts (56%) were transferred in 3 synchronised sows and 2 (66%) became pregnant. In conclusion, after gene editing with programmable nucleases, combining beads-mediated selection with well-designed molecular analyses, we developed a multistep assay that can be used efficiently to detect desired gene edited events in cell colonies suitable for the SCNT. Embryos generated after SCNT were able to establish pregnancies at a high rate.

Targeted RMCE-live piglets generated by SCNT following sequential double site-specific gene modifications of a porcine EGFP line

Program and Abstracts of the 12th Transgenic Technology Meeting (TT2014) The Assembly Rooms, Edinburgh, Scotland, United Kingdom, 6–8 October 2014 The TT2014 Meeting is hosted by: The Roslin Institute, The University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland, United Kingdom. The Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD, Scotland United Kingdom. The Institute of Genetics and Molecular Medicine, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU United Kingdom. 123 Transgenic Res (2014) 23:827–909 DOI 10.1007/s11248-014-9820-1 Springer International Publishing Switzerland 2014