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The implementation of the REACH regulation has imposed the urgent need of developing alternative testing methods to screen large number of compounds more quickly and at lower costs. In this study, a battery of tests, suitable for reproductive toxicology testing, was developed with the objective of detecting the mechanism of action of estrogenic and xenoestrogenic compounds. With this aim, two compounds known for their estrogenic activity, diethylstilbestrol and 17β-estradiol, were used to set up four different in vitro tests: 1) bovine oocyte in vitro maturation assay, 2) bovine preimplantation embryo in vitro culture assay and 3) MCF-7 and 4) BALB/3T3 cell lines proliferation and cytotoxicity assay, respectively. The results show that this battery of tests allows to identify and to distinguish between two major mechanisms of action of (xeno)estrogenic compounds: the receptor-mediated mechanism and the spindle-poison effect on microtubules polimerization.

The in vitro production of porcine embryos is of great interest because of the increasing importance of the swine as an animal model and a tissue donor for biomedical or biotechnological applications. Availability of ovaries at selected time of the day can be a limitation; therefore, the possibility to maintain immature oocytes for some hours can be very useful. The aim of this study is to determine whether holding recovered oocytes at 24°C for 24 h alters the maturation process and/or the developmental capacity. Immature sow oocytes were either matured in vitro for 42 h at 38.5°C (control group; CTR) or kept in 2 mL of HEPES-SOF in the dark at 24°C for 24 h before maturation (experimental group; +24 h). After maturation, cumulus cells were removed, and the number at metaphase II were recorded. For parthenogenetic activation (PGA), oocytes with a visible polar body were activated at 48 h of maturation as previously described (Lagutina et al. 2006). For IVF experiments frozen-thawed boar semen was prepared through a discontinuous density gradient, washed in TALP Ca2+-free, diluted in TALP : SOF = 1 : 1 supplemented with 6 mg mL–1 of fatty acid-free BSA, hypotaurine and epinephrine, mixed with oocytes after partial removal of the cumulus cells, at 43 h of maturation and cultured in 5% CO2 in humidified air at 38.5°C. After 24 h of IVF, oocytes were denuded and cultured in mSOF-1 in atmosphere of 5% O2 and 5% CO2. The same culture conditions were used after parthenogenetic activation. Half of the medium was changed with mSOF-1 at Day 3 and with mSOF-2 at Day 5. The cleavage and the cumulative Day 7 blastocyst (BLD7) rates and cell number of IVF BLD6 were recorded. For each group, blastocysts on Day 6 were fixed and cell number counted, whereas the other embryos were left in culture until Day 7 (cumulative D7 = BLD6 fixed + BLD7). All experiments were done in 3 replicates. The data were compared by Student’s t-test and chi-square test. Maturation rates as recorded for the presence of the polar body did not differ (CTR: 255/312, 82%; +24 h: 208/256, 81%). There was no significant difference (P

For the first time, the N-glycosylation patterns of immunoglobulin G (IgGs) isolated from the serum of two varieties of knockout pigs (lacking N-glycolylneuraminic acid (Neu5Gc) and/or α 1,3 galactose) were examined for the presence of potential glycan xenoantigens and compared to N-glycosylation patterns obtained for wild-type (WT) pig IgGs. Glycopeptide analysis was chosen over glycan release, as protein-A eluates from pig serum may contain IgA and IgM as shown previously. The experiments focused on the analysis of tryptic glycopeptides EEQFNSTYR and AEQFNSTYR from IgGs, and excluded IgA and IgM, in which N-glycosylated peptides have different sequences and masses. WT pig IgG glycopeptides showed the presence of N-glycolylneuraminic acid (Neu5Gc) and absence of N-acetylneuraminic acid (Neu5Ac). Released glycans from the protein-A eluate, however, showed the presence of both types of sialic acids, allowing Neu5Ac to be attributed to IgA and/or IgM. The WT IgG samples also showed the presence of glycans that could by composition have been α-galactosylated, but treatments with α- and β-galactosidases produced inconclusive results as to the linkage nature of the terminal Gal residues. Single knockout (α-Gal transferase) pig IgG was shown to contain Neu5Gc residues, and there was a definite absence of α-Gal. Double knockout pigs (DKO for α-Gal transferase and cytidine monophosphate-A-acetylneuraminic acid hydroxylase (CMAH)) showed the definite absence of α-Gal and Neu5Gc. Instead of the latter, Neu5Ac residues were observed. Further investigation into the sialylation patterns of WT and DKO pig IgGs consisted of esterifying the glycopeptides to allow the detection and differentiation of α-2,3 and α-2,6 sialic acid-galactose linkages. Fucosylation levels were also compared between IgG species.

Higher living standards and better medical care are increasing the lifespan of people around the world. Aging populations, however, have an increased incidence of loss of function or failure of cell, tissue or organ. This has led to the development of new medical disciplines, such as organ transplantation and more recently regenerative medicine. Organ transplantation using human donors (allotransplantation) has made enormous progress thanks to the discovery of novel immunosuppressive drugs. However, the growing demand for organs far exceeds the number of organs potentially available from human donors. Xenotransplantation, namely transplantation between animal donors and man, offers the opportunity to use healthy and highly specialized cells, tissues or solid organs readily available for immediate transfer to patients requiring replacement therapy (Ekser et al., 2012). The therapeutic potential of xenotransplantation is wide, some already in clinical use like bioprosthetic heart valves, decellularized pig tissues (skin, ligaments, bone and cartilage), polyclonal antisera, and pancreatic islet, or in a pre-clinical phase like kidney, heart, liver, lung, cornea, and dopaminergic neurons. The pig is a very suitable species for xenotransplantation for reasons that are well documented in the literature, including physiological and anatomical features, and the availability of a high resolution map of the genome. Moreover, the use of pigs for clinical purposes raises little concern from the wider public, because they are already bred by the millions for meat production worldwide. At present, the use of bioprosthetic heart valves of animal origin is a well established xenotransplantation procedure in clinical practice; however, pig islet xenotransplantation has just entered clinical trials (http://www.lctglobal.com/products/ diabecell/about-type-1-diabetes), and life supporting solid organs transplanted into nonhuman primates still do not survive long enough to warrant implementation of clinical trials (Le Bas-Bernardet et al., 2011) although heterotopic heart transplantation in a primate model has resulted in the remarkable survival of almost three years (Mohiuddin et al., 2015). Therefore, several issues still need to be addressed from the safety point of view, and a number of immunological hurdles have been identified (Table 1) and are currently being addressed at multiple levels (Griesemer et al., 2014). It is expected that the development of novel immunosuppressive strategies for allotransplantation and xenotransplantation, the modification of the immunogenicity of the donor pig through genetic engineering and, possibly the induction of immune tolerance, a phenomenon occasionally observed in allotransplantation, will all contribute to bringing xenotransplantation closer to the clinic (Ekser et al., 2012).

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.

The aim of this study was the development of an alternative testing method based on human embryonic stem cells for prenatal developmental toxicity with particular emphasis on early neural development. To this purpose, we designed an in vitro protocol based on the generation of neural rosettes, representing the in vitro counterpart of the developing neural plate and neural tube, and we challenged this complex cell model with retinoic acid (RA), a well-known teratogenic agent. The cells were exposed to different concentrations of RA during the process of rosettes formation. Morphological and molecular parameters were evaluated in treated as compared with untreated cells to detect both cytotoxicity and specific neural toxicity. Transcriptomic analysis was performed with microarray Affymetrix platform and validated by quantitative real-time PCR for genes relevant to early neural development such as HoxA1, HoxA3, HoxB1, HoxB4, FoxA2, FoxC1, Otx2, and Pax7. The results obtained demonstrated that neural rosette forming cells respond to RA with clear concentration-dependent morphological, and gene expression changes remarkably similar to those induced in vivo, in the developing neural tube, by RA exposure. This strict correspondence indicates that the neural rosette protocol described is capable of detecting specific teratogenic mechanisms causing perturbations of early neural development and therefore represents a promising alternative test for human prenatal developmental toxicity.

Improved sperm selection techniques are needed to increase the efficiency of equine-assisted reproduction. Single layer centrifugation (SLC) of spermatozoa has been shown to improve the quality of stallion sperm samples. In this study, the functionality of selected stallion spermatozoa was tested by intracytoplasmic sperm injection of equine oocytes after selection by SLC through Androcoll-E or by discontinuous density gradient centrifugation (DGC) through Redigrad and Tyrode's medium with added albumin, lactate, and pyruvate. The mean cleavage rates of the injected oocytes from SLC- and DGC-selected spermatozoa were 67% and 66%, respectively, whereas the proportion of blastocysts developing from cleaved oocytes was 28% and 22%, respectively ( P > .05, not significant). An incidental finding was that there was a tendency for SLC-selected spermatozoa to have a higher percentage of spermatozoa with normal morphology than DGC (70% ± 22% vs. 58% ± 38%) and for more blastocysts to be obtained from subfertile ejaculates (21 [19.6%] vs. 15 [14.4%], respectively). In further experiments, stallion spermatozoa bound to hyaluronan, although binding may depend on the semen extender and sperm treatment as well as incubation time. In conclusion, SLC-selected stallion spermatozoa function normally when injected into oocytes. SLC may potentially be better than DGC at selecting spermatozoa from subfertile ejaculates, but this effect needs rigorous investigation with a much larger sample size. Use of the hyaluronan-binding assay for assessing the potential fertility of stallion spermatozoa may be useful but requires further evaluation.

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.

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 genesde novoin 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.

Galli C, Perota A, Brunetti D, Lagutina I, Lazzari G, Lucchini F. Genetic engineering including superseding microinjection: new ways to make GM pigs. Xenotransplantation 2010; 17: 397–410. © 2010 John Wiley & Sons A/S. Abstract: Background: Techniques for genetic engineering of swine are providing genetically modified animals of importance for the field of xenotransplantation, animal models for human diseases and for a variety of research applications. Many of these modifications have been directed toward avoiding naturally existing cellular and antibody responses to species-specific antigens. Methods: A number of techniques are today available to engineering the genome of mammals, these range from the well established less efficient method of DNA microinjection into the zygote, the use of viral vectors, to the more recent use of somatic cell nuclear transfer. The use of enzymatic engineering that are being developed now will refine the precision of the genetic modification combined with the use of new vectors like transposons. Results: The use of somatic cell nuclear transfer is currently the most efficient way to generate genetically modified pigs. The development of enzymatic engineering with zinc-finger nucleases, recombinases and transposons will revolutionize the field. Nevertheless, genetic engineering in large domesticated animals will remain a challenging task. Conclusions: Recent improvements in several fields of cell and molecular biology offer new promises and opportunities toward an easier, cost-effective and efficient generation of transgenic pigs.

In the pig ovary, the transition from primordial to primary and secondary ovarian follicles begins before birth, but antral follicles can be observed, for the first time, at approximately 60-90 d of age. At approximately the same time, secondary follicles become responsive to gonadotropins, leading to the formation of antral follicles. Placing pieces of ovarian tissue under the kidney capsule of immunodeficient (SCID) mice allows the requirements for follicular recruitment and development to be studied. The objective of this study was to investigate if primordial follicles contained in ovarian fragments isolated from newborn piglets (36 +/- 12 h old) and immediately transplanted under the kidney capsule of SCID mice, are able to become responsive to gonadotropins after 60 d (as in an unaltered animal). Ovarian fragments were transplanted under the kidney capsule of three groups of four female and four male SCID mice. The first group did not receive any hormonal treatment for 12 wk. The second group was treated from the 9th week with 1 IU of FSH/LH on alternating days for 3 wk, and the third group was treated with 5 IU Pregnant Mare Serum Ganadotropin (PMSG) 48 h before euthanasia. Primordial follicles contained in ovarian fragments isolated from newborn piglets developed only to the secondary stage. Therefore, development of gonadotropin responsiveness in ovarian fragments xenotransplanted in SCID mice was delayed compared to what occurs in the unaltered animal, and there was minimal response to exogenous gonadotropins.

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 geneNANOGand 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,NANOGwas 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.

The new European chemicals policy for the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) will most probably impose a dramatic increase in the number of animals required for reproductive toxicity testing. For this purpose, the development and validation of alternative methods is urgently needed in order to reduce the use of laboratory animals. The present study describes the inter-laboratory variability and the transferability assessment of an in vitro test able to identify chemical effects during the process of oocyte maturation in a bovine model. The test was developed/optimised within ReProTect, an integrated research project funded by the European Union, joining together 35 partners with complementary expertise in reproductive toxicology. Eight chemicals with well-known toxic properties were tested (benzo[a]pyrene, busulfan, cadmium chloride, cycloheximide, diethylstilbestrol, ketoconazole, methylacetoacetate, mifepristone/RU-486 and DMSO as solvent) on the in vitro maturation (IVM) assay in two well-trained laboratories using the established Standard Operating Procedures. The statistical analysis demonstrated the concordance of results across the laboratories and the reproducibility of the test. We therefore conclude that the IVM test could advance toward the process of validation as alternative in vitro method that, in combination with additional in vitro tests, can become part of an integrated testing strategy in order to predict chemical hazards on mammalian fertility.

Embryonic genome activation (EGA) is a complex process that needs a good orchestration of all biochemical processes at the time of maternal-to-embryonic transition. Mitochondria are strictly dependent on the nucleus for their correct activity as ~1500 mitochondrial genes have nuclear localisation. The finding of transcriptional activation and accumulation of mRNAs related to mitochondrial biogenesis (Mtango et al. 2008 Reprod. Fertil. Dev. 20, 846-859) around the time of EGA confirmed the role of nucleus in this process. Studying mitochondria behaviour in interspecies somatic cell nuclear transfer (iSCNT) embryos (Lagutina et al. 2010 Reproduction 140, 273-285), we have found that at the time of EGA, mitochondria activation could be demonstrated by JC-1 accumulation. We suggested that comparison of the mean green fluorescence intensity (FI) that corresponds to the fluorescence of the monomeric form of the dye and correlates to relative mitochondrial mass (Mancini et al. 1997) in iSCNT and control nuclear transfer (NT) could serve as a test to assess EGA in iSCNT embryos. The aim of this study was to estimate nuclear-cytoplasmic interaction in iSCNT embryos that formed nucleoli at the time of EGA (Lagutina et al. 2011 Reproduction 141, 453-465) such as embryos derived from bovine oocytes and bovine (control), buffalo or ovine donor nuclei, and from porcine oocytes and porcine (control), horse, or rabbit nuclei. Embryos 72 h after activation were stained with 2 μM JC-1 in SOF-HEPES with 10% FCS at 37°C for 1 h. Images were collected using a fluorescein isothiocyanate (FITC) filter and analysed with Adobe Photoshop Elements 2 (Adobe Systems, San Jose, CA, USA). The data are presented as mean FI of the embryo. To demonstrate the effect of EGA inhibition on mitochondria, bovine and porcine NT embryos were cultured in medium supplemented with 25 μg/mL α-amanitin (AA) from 48 to 72 h after activation. The analyses of mean FI of the embryos showed that ovine and buffalo nuclei were able to support mitochondrial mass accumulation in iSCNT embryos with bovine oocytes equal to control bovine NT embryos (35 ± 11.2; 41.9 ± 14.8; 36.2 ± 7.6, respectively) that was significantly higher than in bovine embryos treated with AA (15.4 ± 4.9; P

A previous study from our laboratory (Ramos-Ibeas et al. 2017 Mol. Cell. Biochem. 429, 137-150, 10.1007/s11010-017-2942-z) described the protective effect of sodium pyruvate (SP) against H2O2-induced oxidative stress in somatic and embryonic stem cells. In this study, we tested the same experimental conditions on bovine and swine oocytes during in vitro maturation (IVM). To this aim, cumulus-enclosed oocytes (COC) recovered from bovine and swine ovaries were treated for 1 h with different concentrations of H2O2 and then matured in serum-free TCM-199 with and without SP supplementation. All experiments were done in 3 replicates using 12 to 20 oocytes per experimental group; data were analysed by two-way ANOVA. As a first step, we determined the dose-response curve of H2O2 treatment during IVM, using the reaching of metaphase 2 as endpoint. We found that swine COC were more sensitive to H2O2 damage than bovine COC: indeed, they tolerated a range of concentrations from 128 to 512 μM, whereas the range for bovine COC was between 1024 and 4096 μM. The COC from both species were treated for 1 h with 128 μM (swine) or 2048 μM (bovine) H2O2 and then matured with or without different concentrations of SP. Control COC, unexposed to H2O2, were also matured with and without SP supplementation. We found that the addition of 25 mM SP to swine COC reduced the maturation rate both in control and H2O2-treated groups. Instead, reducing the addition of SP to 10 mM we observed a beneficial effect because the metaphase II level in treated oocytes increased from 38% (15/40) to 67% (24/36) and the difference was statistically significant. In bovine COC, the addition of 25 mM SP did not affect control oocytes during IVM but decreased maturation rate in treated oocytes, whereas 10 mM SP had no effect on the control group and showed a tendency to increase maturation rate in treated oocytes. In a second set of experiments, to determine if cumulus cells acted as a barrier for SP to reach the oocyte, the COC were denuded after H2O2 treatment and matured with or without SP. In swine denuded oocytes (DO) matured with SP, the rate of metaphase II increased significantly both in control [from 71% (34/48) to 92% (44/48)] and in treated groups [from 48% (23/48) to 72% (36/50)]. In bovine DO, the supplementation of SP did not increase the maturation rate significantly (25/41 v. 29/41 in control groups, 12/36 v. 18/40 in treated groups). In conclusion, we found that toxic H2O2 concentrations were approximately 10-fold higher for bovine than for swine, indicating that bovine COC were more resistant to oxidative stress. This could explain why SP supplementation had a significant protective effect in swine but only a minor effect in bovine. Moreover, the difference in SP effect between bovine and swine COC could be due to their morphology because bovine cumulus is often larger and more compact than swine cumulus and this difference could influence how SP reaches the inner oocyte and how it is metabolized. This work was funded by projects EpiHealth FP7 n. 278418, EpiHealthNet FP7-PEOPLE-2012-ITN n. 317146 and Fecund FP7 n. 312097.

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.

The recent vote in the British Parliament allows scientists in principle to create hybrid embryos by transferring human somatic cell nuclei into animal oocytes. This vote opens a fascinating new area of research with the central aim of generating interspecific lines of embryonic stem cells (ESCs) that could potentially be used to understand development, differentiation, gene expression and genomic compatibility. It will also promote human cell therapies, as well as the pharmaceutical industry's search for new drug targets. If this approach is to be successful, many biological questions need to be answered and, in addition, some moral and ethical aspects must be taken into account.

none 5 The cloning of equids was achieved in 2003, several years after the birth of Dolly the sheep and also after the cloning of numerous other laboratory and farm animal species. The delay was because of the limited development in the horse of more classical-assisted reproductive techniques required for successful cloning, such as oocyte maturation and in vitro embryo production. When these technologies were developed, the application of cloning also became possible and cloned horse offspring were obtained. This review summarizes the main technical procedures that are required for cloning equids and the present status of this technique. The first step is competent oocyte maturation, this is followed by oocyte enucleation and reconstruction, using either zona-enclosed or zona-free oocytes, by efficient activation to allow high cleavage rates and finally by a suitable in vitro embryo culture technique. Cloning of the first equid, a mule, was achieved using an in vivo-matured oocytes and immediate transfer of the reconstructed embryo, i.e. at the one cell stage, to the recipient oviduct. In contrast, the first horse offspring was obtained using a complete in vitro procedure from oocyte maturation to embryo culture to the blastocyst stage, followed by non-surgical transfer. Later studies on equine cloning report high efficiency relative to that for other species. Cloned equid offspring reported to date appear to be normal and those that have reached puberty have been confirmed to be fertile. In summary, horse cloning is now a reproducible technique that offers the opportunity to preserve valuable genetics and notably to generate copies of castrated champions and therefore, offspring from those champions that would be impossible to obtain otherwise. Galli C.; Lagutina I.; Duchi R.; Colleoni S.; Lazzari G. Galli C.; Lagutina I.; Duchi R.; Colleoni S.; Lazzari G.

We have shown previously that transgene expression can be suppressed in hematopoietic cells using vectors that are responsive to microRNA (miRNA) regulation. Here we investigate the potential of this approach for more sophisticated control of transgene expression. Analysis of the relationship between miRNA expression levels and target mRNA suppression suggested that suppression depends on a threshold miRNA concentration. Using this information, we generated vectors that rapidly adjust transgene expression in response to changes in miRNA expression. These vectors sharply segregated transgene expression between closely related states of therapeutically relevant cells, including dendritic cells, hematopoietic and embryonic stem cells, and their progeny, allowing positive/negative selection according to the cells' differentiation state. Moreover, two miRNA target sites were combined to restrict transgene expression to a specific cell type in the liver. Notably, the vectors did not detectably perturb endogenous miRNA expression or regulation of natural targets. The properties of miRNA-regulated vectors should allow for safer and more effective therapeutic applications.

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.

The dramatic increase in the number of animals required for reproductive toxicity testing imposes the validation of alternative methods to reduce the use of laboratory animals. As we previously demonstrated for in vitro maturation test of bovine oocytes, the present study describes the transferability assessment and the inter-laboratory variability of an in vitro test able to identify chemical effects during the process of bovine oocyte fertilization. Eight chemicals with well-known toxic properties (benzo[a]pyrene, busulfan, cadmium chloride, cycloheximide, diethylstilbestrol, ketoconazole, methylacetoacetate, mifepristone/RU-486) were tested in two well-trained laboratories. The statistical analysis demonstrated no differences in the EC50 values for each chemical in within (inter-runs) and in between-laboratory variability of the proposed test. We therefore conclude that the bovine in vitro fertilization test could advance toward the validation process as alternative in vitro method and become part of an integrated testing strategy in order to predict chemical hazards on mammalian fertility.

Galactosyl-transferase KO (GalT-KO) pigs represent a potential solution to xenograft rejection, particularly in the context of additional genetic modifications. We have performed life supporting kidney xenotransplantation into baboons utilizing GalT-KO pigs transgenic for human CD55/CD59/CD39/HT. Baboons received tacrolimus, mycophenolate mofetil, corticosteroids and recombinant human C1 inhibitor combined with cyclophosphamide or bortezomib with or without 2-3 plasma exchanges. One baboon received a control GalT-KO xenograft with the latter immunosuppression. All immunosuppressed baboons rejected the xenografts between days 9 and 15 with signs of acute humoral rejection, in contrast to untreated controls (n = 2) that lost their grafts on days 3 and 4. Immunofluorescence analyses showed deposition of IgM, C3, C5b-9 in rejected grafts, without C4d staining, indicating classical complement pathway blockade but alternate pathway activation. Moreover, rejected organs exhibited predominantly monocyte/macrophage infiltration with minimal lymphocyte representation. None of the recipients showed any signs of porcine endogenous retrovirus transmission but some showed evidence of porcine cytomegalovirus (PCMV) replication within the xenografts. Our work indicates that the addition of bortezomib and plasma exchange to the immunosuppressive regimen did not significantly prolong the survival of multi-transgenic GalT-KO renal xenografts. Non-Gal antibodies, the alternative complement pathway, innate mechanisms with monocyte activation and PCMV replication may have contributed to rejection.

The objective of this work was to obtain cloned pig offspring by uterine transfer of blastocysts produced by zona-free manipulation. We started by defining the most suitable culture media for growing pig nuclear transfer embryos produced by zona-free micromanipulation comparing NCSU-23aa with Synthetic Oviduct Fluid (SOFaa) and with in vivo culture in the sheep oviduct. We found that parthenogenetic development to day 7 blastocyst in NCSU-23aa and sheep oviduct was significantly superior as compared to SOFaa (61.8%, 64% and 42.4 respectively) although blastocyst cell number was higher in the latter. Interestingly, when we compared the two media for the culture of nuclear transfer (NT) embryos derived from 3 different donor cell lines, we observed lower rates of development with NCSU-23aa (from 24.5% to 32.4%) while with SOFaa the development was significantly higher for two donor cell lines as compared to the third (44.4%, 48.9% and 20.6% respectively). A total of 244 blastocysts grown in SOFaa were transferred in four synchronized sows on day 5 or 6 of development. Two recipients farrowed 6 and 8 piglets corresponding to an efficiency of development to term of 8% and 16% of the transferred embryos respectively. Eleven pigs are now 10 month of age and those that have reached puberty have been proven to be fertile. Finally, this is the first report on the production of cloned pigs derived from the transfer of NT embryos at the blastocyst stage.

Intracytoplasmic sperm injection (ICSI) is an established method to fertilise equine oocytes, but not all oocytes cleave after ICSI. The aims of the present study were to examine cytoskeleton patterns in oocytes after aging in vitro for 0, 24 or 48 h (Experiment 1) and in potential zygotes that failed to cleave after ICSI of oocytes from donors of different ages (Experiment 2). Cytoplasmic multiasters were observed after oocyte aging for 48 h (P

Chromosomal aberrations are often listed as a significant cause of early embryonic death in the mare, despite the absence of any concrete evidence for their involvement. The current study aimed to validate fluorescent in situ hybridization (FISH) probes to label specific equine chromosomes (ECA2 and ECA4) in interphase nuclei and thereby determine whether numerical chromosome abnormalities occur in horse embryos produced either in vivo (n = 22) or in vitro (IVP: n = 20). Overall, 75% of 36,720 and 88% of 2,978 nuclei in the in vivo developed and IVP embryos were analyzable. Using a scoring system in which extra FISH signals were taken to indicate increases in ploidy and "missing" signals were assumed to be "false negatives," 98% of the cells were scored as diploid and the majority of embryos (30/42: 71%) were classified as exclusively diploid. However, one IVP embryo was recorded as entirely triploid and a further seven IVP and four in vivo embryos were classified as mosaics containing diploid and polyploid cells, such that the incidence of apparently mixoploid embryos tended to be higher for IVP than in vivo embryos (P = 0.118). When the number of FISH signals per nucleus was examined in more detail for 11 of the embryos, the classification as diploid or polyploid was largely supported because 2,174 of 2,274 nuclei (95.6%) contained equal numbers of signals for the two chromosomes. However, the remaining 100 cells (4.4%) had an uneven number of chromosomes and, while it is probable that many were artefacts of the FISH procedure, it is also likely that a proportion were the result of other types of aneuploidy (e.g., trisomy, monosomy, or nullisomy). These results demonstrate that chromosomally abnormal cells are present in morphologically normal equine conceptuses and suggest that IVP may increase their likelihood. Definitive distinction between polyploidy, aneuploidy and FISH artefacts would require the use of more than one probe per chromosome and/or probes for more than two chromosomes.

International audience; In contrast to mice, in sheep no genome-wide demethylation of the paternal genome occurs within the first postfertilization cell cycle. This difference could be due either to an absence of a sheep demethylase activity that is present in mouse ooplasm or to an increased protection of methylated cytosine residues in sheep sperm. Here, we use interspecies intracytoplasmic sperm injection to demonstrate that sheep sperm DNA can be demethylated in mouse oocytes. Surprisingly, mouse sperm can also be demethylated to a limited extent in sheep oocytes. Our results suggest that the murine demethylation process is facilitated either by a sperm-derived factor or by male pronuclear chromatin composition.

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. developmental biology, early development, embryo, implantation, in vitro fertilization

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.

Blastocyst formation rates during horse embryo in vitro production (IVP) are disappointing, and embryos that blastulate in culture fail to produce the characteristic and vital glycoprotein capsule. The aim of this study was to evaluate the impact of IVP on horse embryo development and capsule formation. IVP embryos were produced by intracytoplasmic sperm injection of in vitro matured oocytes and either culture in synthetic oviduct fluid (SOF) or temporary transfer to the oviduct of a ewe. Control embryos were flushed from the uterus of mares 6-9 days after ovulation. Embryo morphology was evaluated with light microscopy, and multiphoton scanning confocal microscopy was used to examine the distribution of microfilaments (AlexaFluor-Phalloidin stained) and the rate of apoptosis (cells with fragmented or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive nuclei). To examine the influence of culture on capsule formation, conceptuses were stained with a monoclonal antibody specific for capsular glycoproteins (OC-1). The blastocyst rate was higher for zygotes transferred to a sheep's oviduct (16%) than for those cultured in SOF (6.3%). Day 7 IVP embryos were small and compact with relatively few cells, little or no blastocoele, and an indistinct inner cell mass. IVP embryos had high percentages of apoptotic cells (10% versus 0.3% for in vivo embryos) and irregularly distributed microfilaments. Although they secreted capsular glycoproteins, the latter did not form a normal capsule but instead permeated into the zona pellucida or remained in patches on the trophectodermal surface. These results demonstrate that the initial layer of capsule is composed of OC-1-reactive glycoproteins and that embryo development ex vivo is retarded and aberrant, with capsule formation failing as a result of failed glycoprotein aggregation.

We have established a transcription map of individual bovine embryos using semiquantitative reverse transcriptase-polymerase chain reaction to detect the levels of six marker genes involved in early embryo differentiation. The critical step of compaction during preimplantation development is often not accomplished or it takes place for only a short period in in vitro generated embryos, which may result in reduced viability. Compaction is accompanied by the assembly of intercellular tight junctions (TJs) as a barrier against the extraembryonic environment and as a prerequisite for blastocele formation. In the present study, we have related the expression of TJ gene mRNA in individual bovine embryos to their developmental stage, their competence to undergo a clear period of compaction before blastocyst formation, and their in vitro or in vivo origin. Our results indicate that embryos that showed a detectable and well-formed compaction period in vitro are of similar quality to their in vivo counterparts. Starting from the same amount of maternal message, in vivo and in vitro development differ most during the critical period of the major switch from maternal to embryonic genomic control before a dramatic increase of TJ mRNAs occurs upon blastocyst formation. Failure to compact in vitro results in significant reduction of specific transcript levels, in a manner that depends on culture conditions, which may contribute to reduced viability. We conclude that TJ mRNA expression levels are sensitive to environmental conditions that may influence the developmental potential of bovine blastocysts.

Francesca Caloni 1, Patrizia Costa 2, Gianni Dal Negro 3, Isabella De Angelis 4, Giovanna Lazzari 5, Giacomo Metteucci 6, and Augusto Vitale 7 Universita degli Studi di Milano, Department of Health, Animal Science and Food Safety, Milan, Italy; Universita degli Studi di Milano, laboratory Animal Science and Medicine Specialization School, Milan, Italy; GlaxoSmithKline RD 4 Istituto Superiore di Sanita, Department of environment and Primary Prevention, Rome, Italy; Avantea, laboratory of Reproductive technologies, Cremona, Italy; Universita degli studi di Milano, lAS and Animal Welfare Consultant, Milan, Italy; Istituto Superiore di Sanita, Department of Cell Biology and Neuroscience, Section of Behavioural Neuroscience, Rome, Italy

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.

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.

The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily that binds a broad repertoire of advanced glycation end products ligands. It is constitutively expressed at a high level during embryonic development, but its levels decrease in adult tissues. The RAGE-ligand interaction induces a series of signal transduction cascades and leads to the activation of several inflammatory signalling pathways. This is why RAGE is frequently associated with pro-inflammatory responses and it is implicated as an underlying condition in immune disorders, cardiovascular diseases, diabetes, Alzheimer’s disease, and cancer. However, the physiological function of RAGE during embryogenesis and in aging is largely undefined. The aim of this work was to determine the basal levels on RAGE expression in bovine oocytes (germinal vesicle, GV, and in vitro-matured metaphase II) and in vitro-derived embryos developed in SOF medium, by qPCR (3 replicates each containing pooled RNA from 20 oocytes or embryos, t-test P

Although it was demonstrated that a-1,3 galactosyltransferase knock out (Gal-KO) pig organs transplanted into primates were protected from hyperacute rejection, hCD55 expression in donors is considered necessary to protect cells and tissues from complement-mediated damage. To provide sufficient number of cloned transgenic animals with high expression of CD55 for xenotransplantation, we combined intensive screening of colonies (ICC and WB) with

Somatic cell nuclear transfer (SCNT) was initially developed in livestock for the purpose of accelerating the widespread use of superior genotypes. Many problems still exist now after 15 years of research due to the limited understanding of genome reprogramming. However, SCNT has proved to be a powerful tool to make copies of selected individuals in different species, and to study pluripotency and differentiation, opening new avenues of research in regenerative medicine and representing the main route for making transgenic livestock. In equines, the possibility of making copies of outstanding individuals, for many different reasons, has found its natural application in the generation of animals to be used as breeding stock. The low incidence of abnormalities observed in this species makes the technique more attractive to horse breeders. The extent of application of equine cloning will also depend on regulations that will be dictated by the breed registries, although in recent years the attitude towards cloning has become more positive.

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.

Bovine herpesvirus 4 (BoHV-4) is a gamma herpesvirus with no clear disease association. Previous studies have demonstrated that macrophages can harbour persistent BoHV-4. Since mesenchymal stem cells in bone marrow regulate the differentiation and proliferation of adjacent haematopoietic precursors, such as macrophages, the interaction between BoHV-4 and mesenchymal stem cells was investigated. Primary bovine mesenchymal stem cells were highly permissive to support full replication of BoHV-4. This finding could be considered a new important step in studies on the potential pathogenesis related to BoHV-4.

In the pig, the efficiency of in vitro embryo production and somatic cell nuclear transfer (SCNT) procedures remains limited. It has been suggested that prematuration treatments (pre-IVM) based on the prolongation of a patent, bidirectional crosstalk between the oocyte and the cumulus cells through gap junction mediate communication (GJC), with the maintenance of a proper level of cAMP, could improve the developmental capability of oocytes. The aim of this study was to assess: 1) dose-dependent effects of cilostamide on nuclear maturation kinetics, 2) the relationship between treatments on GJC functionality and large-scale chromatin configuration changes, and 3) the impact of treatments on developmental competence acquisition after parthenogenetic activation (PA) and SCNT. Accordingly, cumulus-oocyte complexes were collected from 3- to 6-mm antral follicles and cultured for 24 h in defined culture medium with or without 1 μM cilostamide. GJC functionality was assessed by Lucifer yellow microinjection, while chromatin configuration was evaluated by fluorescence microscopy after nuclear staining. Cilostamide administration sustained functional coupling for up to 24 h of culture and delayed meiotic resumption, as only 25.6% of cilostamide-treated oocytes reached the pro-metaphase I stage compared to the control (69.7%; P < 0.05). Moreover, progressive chromatin condensation was delayed before meiotic resumption based upon G2/M biomarker phosphoprotein epitope acquisition using immunolocalization. Importantly, cilostamide treatment under these conditions improved oocyte developmental competence, as reflected in higher blastocyst quality after both parthenogenetic activation and SCNT.

Induction of neural differentiation from embryonic pluripotent stem cells (ES/EpiSc), both of mouse and primates, has been extensively published by several research teams. However, direct derivation of organized neuroectoderm in vitro from blastocyst stage embryos of rodents or primates has not been reported so far. Here we describe a method of direct neural differentiation from the inner cell mass cells of preimplantation bovine embryos, without the intermediate step of ES/EpiSc cells derivation (Lazzari et al., Stem cells 24:2514-2521, 2006). Proliferating neural precursors cells lines, and both central and peripheral nervous system derivatives, can be obtained providing a unique in vitro model of early neurulation events in mammals.

In the pig, the efficiency of in vitro embryo production and somatic cell nuclear transfer (SCNT) procedures remains limited. It has been suggested that prematuration treatments (pre-IVM) based on the prolongation of a patent, bidirectional crosstalk between the oocyte and the cumulus cells through gap junction mediate communication (GJC), with the maintenance of a proper level of cAMP, could improve the developmental capability of oocytes. The aim of this study was to assess: 1) dose-dependent effects of cilostamide on nuclear maturation kinetics, 2) the relationship between treatments on GJC functionality and large-scale chromatin configuration changes, and 3) the impact of treatments on developmental competence acquisition after parthenogenetic activation (PA) and SCNT. Accordingly, cumulus-oocyte complexes were collected from 3- to 6-mm antral follicles and cultured for 24 h in defined culture medium with or without 1 μM cilostamide. GJC functionality was assessed by Lucifer yellow microinjection, while chromatin configuration was evaluated by fluorescence microscopy after nuclear staining. Cilostamide administration sustained functional coupling for up to 24 h of culture and delayed meiotic resumption, as only 25.6% of cilostamide-treated oocytes reached the pro-metaphase I stage compared to the control (69.7%; P0.05). Moreover, progressive chromatin condensation was delayed before meiotic resumption based upon G2/M biomarker phosphoprotein epitope acquisition using immunolocalization. Importantly, cilostamide treatment under these conditions improved oocyte developmental competence, as reflected in higher blastocyst quality after both parthenogenetic activation and SCNT.

Assisted reproductive techniques developed for cattle in the last 25 years, like ovum pick up (OPU), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer, have been transferred and adapted to buffalo and horses. The successful clinical applications of these techniques require both the clinical skills specific to each animal species and an experienced laboratory team to support the in vitro phase of the work. In cattle, OPU can be considered a consolidated technology that is rapidly outpacing conventional superovulation for embryo transfer. In buffalo, OPU represents the only possibility for embryo production to advance the implementation of embryo-based biotechnologies in that industry, although it is still mainly in the developmental phase. In the horse, OPU is now an established procedure for breeding from infertile and sporting mares throughout the year. It requires ICSI that in the horse, contrary to what happens in cattle and buffalo, is very efficient and the only option because conventional IVF does not work. Somatic cell nuclear transfer is destined to fill a very small niche for generating animals of extremely high commercial value. The efficiency is low, but because normal animals can be generated it is likely that advancing our knowledge in that field might improve the technology and reduce its cost.

Induction of neural differentiation from embryonic pluripotent stem cells (ES/EpiSc), both of mouse and primates, has been extensively published by several research teams. However, direct derivation of organized neuroectoderm in vitro from blastocyst stage embryos of rodents or primates has not been reported so far. Here we describe a method of direct neural differentiation from the inner cell mass cells of preimplantation bovine embryos, without the intermediate step of ES/EpiSc cells derivation (Lazzari et al., Stem cells 24:2514-2521, 2006). Proliferating neural precursors cells lines, and both central and peripheral nervous system derivatives, can be obtained providing a unique in vitro model of early neurulation events in mammals.

The present situation in Europe can be described as the acceptance of the "precautionary" principle, by which any new biotech application is considered hazardous until proven safe beyond any possible doubt. This attitude is now so well established in the public opinion that it is very difficult for people to accept products (food in particular) that have been developed through biotech. People prefer traditional products, not because they are safer but because they were always there. For the reasons mentioned above, de facto there is no one significant European regulation on cloning, rather, every country has its own approach or variations.