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Cesare Galli and Giovanna Lazzari

SCNT (somatic cell nuclear transfer) has complemented the toolbox of ARTs offering yet another technique to reproduce animals in an unprecedented way. Despite remarkable achievements, SCNT suffers low efficiency, high pregnancy losses and higher than normal stillbirth rates that makes it an expensive technique to reproduce animals. Moreover, due to welfare issues associated with gestation and the newborn offspring, it is banned in some countries. It has become evident that these problems are of epigenetic nature associated with incomplete genome reprogramming, observed more frequently in ruminants and less often and of minor degree in pigs and horses. Genome editing is enormously benefiting from SCNT to turn genome edited cells into animals, even if zygote microinjection of CRISPR/Cas9 will become an alternative route in some occasions. SCNT will also be a route to reprogram somatic cell to pluripotency since bona fide iPSC in livestock are missing while embryonic stem cells have been now established. This opens the way to other technologies like the development of artificial gametes or interspecies nuclear transfer. To strengthen its commercial applications, SCNT will face three major challenges, that is, intellectual property (extremely unclear in genome editing), regulatory approval by the relevant authorities of the resuting potential products and finally, acceptance by the public who will eventually decide with its behavior the life or the death of the technology.

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Irina Lagutina, Valeri Zakhartchenko, Helena Fulka, Silvia Colleoni, Eckhard Wolf, Josef Fulka Jr, Giovanna Lazzari, and Cesare Galli

The most successful development of interspecies somatic cell nuclear transfer (iSCNT) embryos has been achieved in closely related species. The analyses of embryonic gene activity in iSCNT embryos of different species combinations have revealed the existence of significant aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). However, there are many studies with successful blastocyst (BL) development of iSCNT embryos derived from donor cells and oocytes of animal species with distant taxonomical relations (inter-family/inter-class) that should indicate proper EGA at least in terms of RNA polymerase I activation, nucleoli formation, and activation of genes engaged in morula and BL formation. We investigated the ability of bovine, porcine, and rabbit oocytes to activate embryonic nucleoli formation in the nuclei of somatic cells of different mammalian species. In iSCNT embryos, nucleoli precursor bodies originate from the oocyte, while most proteins engaged in the formation of mature nucleoli should be transcribed from genes de novo in the donor nucleus at the time of EGA. Thus, the success of nucleoli formation depends on species compatibility of many components of this complex process. We demonstrate that the time and cell stage of nucleoli formation are under the control of recipient ooplasm. Oocytes of the studied species possess different abilities to support nucleoli formation. Formation of nucleoli, which is a complex but small part of the whole process of EGA, is essential but not absolutely sufficient for the development of iSCNT embryos to the morula and BL stages.

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Irina Lagutina, Helena Fulka, Tiziana A L Brevini, Stefania Antonini, Dario Brunetti, Silvia Colleoni, Fulvio Gandolfi, Giovanna Lazzari, Josef Fulka Jr, and Cesare Galli

The best results of inter-species somatic cell nuclear transfer (iSCNT) in mammals were obtained using closely related species that can hybridise naturally. However, in the last years, many reports describing blastocyst development following iSCNT between species with distant taxonomical relations (inter-classes, inter-order and inter-family) have been published. This indicates that embryonic genome activation (EGA) in xeno-cytoplasm is possible, albeit very rarely. Using a bovine–pig (inter-family) iSCNT model, we studied the basic characteristics of EGA: expression and activity of RNA polymerase II (RNA Pol II), formation of nucleoli (as an indicator of RNA polymerase I (RNA Pol I) activity), expression of the key pluripotency gene NANOG and alteration of mitochondrial mass. In control embryos (obtained by IVF or iSCNT), EGA was characterised by RNA Pol II accumulation and massive production of poly-adenylated transcripts (detected with oligo dT probes) in blastomere nuclei, and formation of nucleoli as a result of RNA Pol I activity. Conversely, iSCNT embryos were characterised by the absence of accumulation and low activity of RNA Pol II and inability to form active mature nucleoli. Moreover, in iSCNT embryos, NANOG was not expressed, and mitochondria mass was significantly lower than in intra-species embryos. Finally, the complete developmental block at the 16–25-cell stage for pig–bovine iSCNT embryos and at the four-cell stage for bovine–pig iSCNT embryos strongly suggests that EGA is not taking place in iSCNT embryos. Thus, our experiments clearly demonstrate poor nucleus–cytoplasm compatibility between these animal species.

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Irina Lagutina, Giovanna Lazzari, Roberto Duchi, Silvia Colleoni, Nunzia Ponderato, Paola Turini, Gabriella Crotti, and Cesare Galli

The objective of the present work was to investigate and clarify the factors affecting the efficiency of somatic cell nuclear transfer (NT) in the horse, including embryo reconstruction, in vitro culture to the blastocyst stage, embryo transfer, pregnancy monitoring and production of offspring. Matured oocytes, with zona pellucida or after zona removal, were fused to cumulus cells, granulosa cells, and fetal and adult fibroblasts, and fused couplets were cultured in vitro. Blastocyst development to Day 8 varied significantly among donor cells (from 1.3% to 16%, P < 0.05). In total, 137 nuclear transfer-embryos were transferred nonsurgically to 58 recipient mares. Pregnancy rate after transfer of NT-embryos derived from adult fibroblasts from three donor animals was 24.3% (9/37 mares transferred corresponding to 9/101 blastocysts transferred), while only 1/18 (5.6%) of NT-blastocysts derived from one fetal cell line gave rise to a pregnancy (corresponding to 1/33 blastocysts transferred). Overall, seven pregnancies were confirmed at 35 days, and two went to term delivering two live foals. One foal died 40 h after birth of acute septicemia while the other foal was healthy and is currently 2 months old. These results indicate that (a) the zona-free method allows high fusion rate and optimal use of equine oocytes, (b) different donor cell cultures have different abilities to support blastocyst development, (c) blastocyst formation rate does not correlate with pregnancy fate and (d) healthy offspring can be obtained by somatic cell nuclear transfer in the horse.

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Roberto Puglisi, Roberta Vanni, Andrea Galli, Donatella Balduzzi, Katia Parati, Graziella Bongioni, Gabriella Crotti, Roberto Duchi, Cesare Galli, Giovanna Lazzari, and Riccardo Aleandri

The methodologies used for cytometric sorting of fresh spermatozoa never allowed a clear resolution of sexual chromosomes of frozen–thawed semen. To devise a novel method for the production of bovine predefined sexed embryos using frozen–thawed semen, sorting efficiency of different protocols was studied using a new quantitative real-time PCR method to verify the purity of sexed semen. To this aim, after Percoll separation, frozen–thawed samples were stained at different temperatures and concentrations of Hoechst 33342 using a short-incubation time. The concentration of Hoechst 33342 of 500 μg/ml at a temperature of 37 °C provided good and stable fluorescence signals. Preventing the sperm clustering by adding 0.6% BSA in the 90% Percoll fraction led to X-bearing sperms purity of 91±2%. Thereafter, sorted sperms were used for in vitro fertilisation (IVF). Despite the lower cleavage rates reported in the sorted groups when compared with the control groups (40 vs 48%, P<0.01), blastocyst formation in the sorted and control groups was not different (27 vs 24% of the cleaved respectively). The PCR analysis of 30 blastocysts confirmed 26 embryos to be correctly sexed (87%). Transfer of two embryos per recipient into six synchronised heifers resulted in four pregnancies. Two abortions occurred at day 60, while two pregnancies went to term delivering two female calves. In conclusion, high purity and repeatabilityof sorting was obtained with frozen–thawed bull semen that was subsequently used for IVF giving rise toviable embryos and offspring. In addition, real-time PCR revealed to be an optimal support for these studies, providing a rapid and reliable estimation of flowcytometric efficiency.