421 PRODUCTION AND CHARACTERIZATION OF TRANSGENIC BOVINE EMBRYOS OBTAINED BY INTRACYTOPLASMIC SPERM INJECTION-MEDIATED GENE TRANSFER ASSISTED BY DIFFERENT CHEMICAL ACTIVATION TREATMENTS

Pronuclear microinjection and intracytoplasmic sperm injection-mediated gene transfer (ICSI-mgt) are useful techniques to obtain transgenic animals. Nevertheless, a high frequency of mosaic expression is observed in embryos and offspring produced by these techniques. A possible explanation is that the transgene integrates in the embryo genome after the first cell division. Our main objective was to develop a new technique to generate transgenic bovine embryos without mosaic expression and with high efficiency. We hypothesize that fertilizing metaphase II (MII) oocytes with transgenic androgenetic haploidblastomeres (AHB) (from mosaic embryos) would result in non-mosaic transgenic embryos. To this aim, in the first experiment we generated AHB by enucleating IVM MII oocytes, before or after injecting with a single spermatozoon incubated with pCX-EGFP (enhanced green fluorescent protein) plasmid. These treatments were analyzed by Fisher test (P < 0.05). The rate of cleavage of the androgenetic transgenic embryos enucleated before and after ICSI-mgt was 35.1% (34/97) and 61.2% (71/116), respectively (P < 0.05). These embryos showed expression of EGFPof 11.8% (4/34) and 42.3% (30/71) (P < 0.05) with 0% (0/34) and 9.9% (7/71) of non-mosaic expression. The haploid condition of the androgenetic embryos was confirmed by karyotype analysis. After this first approach, we chose the procedure of enucleation after ICSI for successive experiments. In the second experiment, the haploid androgenetic embryos (4 to 16 cells) were disaggregated, and the AHB obtained were used to fertilize MII oocytes. Fertilization was carried out by fusing a single AHB to a zona-free MII oocyte, followed by chemical activation. Presumptive zygotes were cultured in SOF medium in the well of the well (WOW) system. To confirm fertilization, single AHB produced with sexed Y spermatozoa and embryos generated with them were checked by PCR using Y- and X-specific sequence primers. PCR analysis confirmed Y-specific sequences in all the AHB and XY-specific sequences in each of the analyzed embryos. FISH analysis on blastocysts was performed with a specific probe for a Y chromosome sequence, confirming the sexed sperm genome in all blastocyst cells. Additionally, the expression pattern of Oct-4 (pluripotent marker gene) was examined in the blastocysts by inmunocytochemistry with a confocal microscope. Blastocysts displayed a pattern of Oct-4 expression similar to that of IVF embryos, indicating efficient nuclear reprogramming. Finally, we fertilized MII oocytes with EGFP-AHB to produce transgenic bovine embryos without mosaic expression. The development reached 85.1% of cleavage and 9.0% of blastocysts (n = 84). One hundred percent of the embryos showed EGFP expression, with 90.1% non-mosaic expression. In conclusion, our results proved that it is possible to use AHB for fertilization of MII oocyte, and that fertilization with transgenic AHB is a highly efficient technique for the generation of transgenic non-mosaic bovine embryos.

Transcription activator-like effector nucleases (TALENs) are highly efficient genome-editing tools that facilitate precise site-directed modifications by inducing double-strand breaks in the host genome, enabling targeted gene knock-in or knock-out. The Rosa26 locus has been widely recognized as a safe harbor for gene integration across various species. This study aimed to assess the quantitative expression of pluripotency-related genes in transgenic goat embryos produced via hand-guided cloning, using goat fetal fibroblast cells with a GFP gene integrated at the Rosa26 locus via TALEN-mediated genome editing. Goat fetal fibroblast cells were co-transfected with a donor vector carrying the GFP gene and TALEN expression vectors. Transgenic goat embryos were generated using GFP-expressing transfected cells as nuclear donors for hand-guided cloning. The early-stage developmental rates of transgenic embryos were comparable to those of non-transgenic (control) embryos. Quantitative PCR (qPCR) analysis revealed no significant differences (P< 0.05) in the relative expression of pluripotency markers OCT4 (0.996±0.298 vs. 1.00) and NANOG (0.744±0.354 vs. 1.00) between transgenic and non-transgenic cloned embryos. These findings indicate that TALEN-mediated transfection did not impair the regenerative capacity of donor cells, allowing for normal embryonic development comparable to non-transgenic embryos. Thus, TALENs represent a promising tool for targeted gene insertion in the production of site-specific transgenic embryos.

Production of transgenic mouse embryos by microinjection is a well established and successful technique. However, when microinjection protocols were used for bovine, the amount of the oocyte lipid content did not allow the production of bovine transgenic embryos. Sperm-mediated gene transfer (SMGT) is an alternative for this species because it has lower cost and does not require microinjection handling. One of the procedures to introduce exogen DNA into oocytes is by means of sperm capacitated with calcium ionophore (CaI). The aim of this work was to evaluate different CaI concentrations ([CaI]), sperm incubation times with CaI (tCa), and incubation times of sperm capacitated with DNA (tDNA) (EYFP; Clontech, Palo Alta, CA, USA) to establish a satisfactory method for IVP of bovine transgenic embryos. Slaughterhouse oocytes with compact cumulus and uniform ooplasm were in vitro maturated in TCM-199 medium + 10% FCS + FSH + hCG + estradiol (E2) + piruvate + gentamicin under 5% CO2 in air, at 39�C and high humidified atmosphere for 24 h. Semen was thawed in a water bath at 37�C for 30 s and separated by Percoll gradient (45/90%) at 600g for 30 min. After this procedure, sperm cells were washed in TALP-semen medium by centrifugation at 200g for 5 min at room temperature. Supernatant was removed and capacitation (5 � 106 spermatozoa/group) was induced with CaI (250 nM or 500 nM for 1 or 5 min). Capacitated sperm cells were incubated with 500 ng/mL DNA for 1 or 2 h. Nontreated spermatozoa were used as control group. Sperm cells (1 � 105) were used to inseminate 20 oocytes/90 mL microdroplets for 18 h. The presumptive zygotes were co-cultured in SOFaa medium with a granulosa cell monolayer under high humidified atmosphere, at 39�C and 5% CO2 in air. Blastocyst rates were analyzed by ANOVA. Independent variables were replicate, [CaI], tCa, tDNA, and the double and triple interactions among the last three variables; when appropriate, means were compared by orthogonal contrasts. There was [CaI] � tCa � tDNA interaction for blastocyst rate (P &lt; 0.02). Treatments with 250 nM ([CaI]), 5 min (tCaI), and 1 h (tDNA) or 500 nM ([CaI]), 1 min (tCaI), and 1 h (tDNA) resulted in 36.1% and 37.4% blastocyst rates, respectively, similar to the control group (30.5%; P &gt; 0.4). These results demonstrated that it is possible to capacitate spermatozoa with CaI to produce transgenic embryos, without alteration of blastocyst rate. This work was supported by FAPESP 03/08542-5 and 03/07456-8.

Diabetes is a growing disease worldwide and has emerged as a major healthcare problem in India. Insulin is an essential medicine for the treatment of diabetes. Large dairy animals, such as buffaloes and cows, may be used as bioreactors for cost-effective production of human insulin. The present study was aimed to produce transgenic buffalo embryos containing the human insulin gene through hand-guided cloning for production of transgenic animals. Buffalo female fetal fibroblast cells at passage number 3 were transfected using mammary gland- specific expression vector containing the human insulin gene under buffalo β-lactoglobulin promoter by nucleofection method and cultured with G418 drug for 3 weeks to obtain positive transgenic cell clones. Transgene integration into buffalo female fetal fibroblast genome was confirmed by PCR and Southern blotting. Nontransfected and transgene integrated cells were used as nuclear donors to produce embryos by the hand-guided cloning technique. The developmental competence and quality of embryos as judged by total cell number and TUNEL assay were compared among transgenic and nontransgenic (control) embryos. The blastocyst rate was lower (P &lt; 0.05) for transgenic embryos than that of nontransgenic cloned embryos (35.97 ± 2.16 v. 45.80 ± 4.11, respectively). The apoptotic index was found to be lower (P &lt; 0.05) for control blastocysts than that for transgenic blastocysts. However, the total cell number was similar (P &lt; 0.05) among transgenic and control cloned blastocysts. Thus, transgenic cells, and subsequently transgenic embryos containing the human insulin gene, were successfully produced and transferred in recipients. In the future, these may be used for production of transgenic buffalo expressing human insulin in its milk and thus can be further utilised in large-scale production of human insulin.

In the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1 (IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasts cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h. Parthenogenetic ooctyes were used as a model to investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO-Faa and CR1aa; 86.3% vs 83.9%, P>0.05 and 23.1% vs 17.2%, P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P>0.05). After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly expressing red fluorescence. Two of the red fluorescent blastocysts were randomly selected to identify transgene by polymerase chain reaction. Both were positive. These results showed that: (i) RFP and Neo ( r ) genes were correctly expressed indicating that transgenic somatic cell lines and positive transgenic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and optimization, transgenic cashmere goat embryos expressing red fluorescence and containing an IGF1 expression cassette were obtained, which was sufficient for production of transgenic cashmere goats.

Comparative analysis of buffalo (Bubalus bubalis) non-transgenic and transgenic embryos containing human insulin gene, produced by SCNT

ICSI-mediated gene transfer is a powerful technique used to produce transgenic mice and pigs. However, this method of transgenesis has not been applied in bovine due to low embryo development, which is presumed to be a consequence of a failure in sperm factor delivery after ICSI in this species. To bypass this problem, we assisted ICSI with chemical activation, employing two Ionomycin (Io) exposures and 6-Dimethylaminopurine (DMAP) or a novel drug, Dehydroleucodine (DhL). Cumulus–oocyte complexes were aspirated from ovaries obtained from a local slaughterhouse and in vitro matured in bicarbonate-buffered TCM-199 containing 10% FBS, 10 μg mL–1 FSH, 0.3 mm sodium pyruvate, 100 μm cysteamine and 10 UI mL–1 penicillin. IVM conditions were 6% CO2 in humidified air at 39°C for 24 h. MII oocytes were selected and used immediately for ICSI. Sperm samples were frozen/thawed by standard procedures. Coincubation of spermatozoa with DNA construction (pCX-EGFP) was carried out in Na citrate 2.8%, with 0.5 μg plasmid million–1 spermatozoa for 5 min at 0°C. Then, spermatozoa were used for ICSI. Injected oocytes were activated in 5 μm Io for 4 min and placed in TCM-199 for 3 h to allow second polar body emission. Afterwards, some of the oocytes were subjected to a second exposure of Io. Oocytes exposed once or twice to Io were then incubated with 2 mm DMAP (groups Io-DMAP and 2Io-DMAP) or 5 mm DhL (groups Io-DhL and 2Io-DhL) for 3 h. Control groups (Io and 2Io) were not treated with DMAP or DhL. Embryos were cultured in the IVM droplets. EGFP expression was daily evaluated in fluorescence microscope under blue light (488 nm). Significant differences between groups were evaluated by Fisher test (Table 1). DhL chemical activation improved neither development nor transgenesis rates. The double Io exposure significantly improved embryo development. The second exposure to Io previous chemical activation with DMAP resulted in an increase in the percentage of EGFP-expressing embryos. Our results indicate that activation with double Io-DMAP could be considered an alternative assistance for ICSI mediated gene transfer in bovine. Table 1.Effect of activation assisting transgenic ICSI on development and expression of bovine embryos

Data from mice showed that follistatin can block myostatin activity in vivo and follistatin overexpressed in transgenic mice can induce dramatic increases in muscle mass. In order to determine whether this effect of follistatin have the same results in swine, we design experiment to make a follistatin transgenic porcine by somatic cell nuclear transfer. We hope to get a follistatin transgenic porcine that has double muscle mass. The present study aimed to construct follistatin transgenic embryos by somatic cell nuclear transfer. A follistatin (FST) expression vector, pFST-1, which contains a porcine FST cDNA (with modification of codon usage) driven by the human cytomegalovirus (CMV) promoter, was constructed. A SV40-neo expression cassette as a selection marker was inserted into pFST-1 to generate pST101, which was transfected into early-passage male primary porcine fetal fibroblast cells by the lipofection method; the transfected cells were selected with 400 mg mL–1 G418. The G418-resistant colonies were pooled. Cell genome DNA was extracted and PCR analysis showed that CMV promoter, follistatin CDS, and SV40 polyA can be detected, indicating that the expression construct had integrated in cell genome. RT-PCR analysis showed that pST101 cells contained higher amounts of FST cDNA compared with the non-transfected cells, indicating that the follistatin was overexpressed in the primary porcine cells. Then, pST101 cell as donor cell and non-transfected cells as control donor cell were used to construct somatic cell nuclear transfer (SCNT) embryos. All of these SCNT embryos were cultured in Porcine Zygote Medium-3. The cleavage rate and blastocyst rate was assessed on 48 h and Day 6. All data were subjected to a Generalized Linear Model Procedure (PROC-GLM) of Statistical Analysis System (SAS, SAS Institute Inc., Cary, NC, USA). The cleavage rate of pST101 cell and control cell derived SCNT embryos were 83.6 ± 2.0% (267/318) and 84.0 ± 2.2% (139/167) respectively, there were no significant (P &gt; 0.05) differences between them. The blastocyst rate from pST101 cell and control cell derived SCNT embryos were 21.4 ± 1.8% (65/318) and 15.0 ± 3.0% (26/167) respectively, the SCNT embryos derived from pST101 cell showed a higher (P &lt; 0.05) blastocyst rate. These results indicated that follistatin transgenic porcine embryo can be successfully constructed and can develop in vitro to blastocyst with a higher rate than control SCNT embryo. Further study will be focused on the full development in vivo of the transgenic embryos and phenotype of transgenic piglets. This work was supported in part by the National High-tech Research and Development Program of China (2008AA101006) and Major Projects for Transgenic Animals of China (2008ZX08006-002).

Nuclear transfer (NT) is associated with epigenetic reprogramming of donor cells. Expression of certain genes in these cells might facilitate their expression in the NT embryo. This research was aimed to investigate the effect of constitutive expression of OCT4 in bovine somatic cells used for NT on the developmental potential of derived cloned embryos as well as in the expression of pluripotency markers in the Day-7 resulting embryos. Cloned blastocysts were generated from five cell lines that expressed OCT4. Pools of blastocysts were screened to detect OCT4, SOX2, and NANOG by qPCR. In vitro-fertilized time-matched blastocysts were used as controls. The development potential was assessed on the basis of blastocysts rate; grading and total cell counts at Day 7. OCT4 expression in the cell lines positively correlates with blastocysts rate (r = 0.92; p = 0.02), number of grade I blastocysts (r = 0.96; p = 0.01), and total cell number (r = 0.98; p = 0.002). The high expression of OCT4 in the cell line did not improve the final outcome of cloning. Somatic expression of OCT4 lead to increased expression of OCT4 and SOX2 in cloned grade I blastocysts; however, there was a bigger variability in OCT4 and SOX2 (p = 0.03; p = 0.02) expression in the embryos generated from cells expressing highest levels of OCT4. Probably the higher variability in OCT4 expression in cloned embryos is due to incorrect reprogramming and incapability of the oocyte to correct for higher OCT4 levels. For that reason, we concluded that OCT4 expression in somatic cells is not a good prognosis marker for selecting cell lines.

The Oct4 gene is crucial for undisturbed early embryonic development and maintenance of pluripotency in the mouse. It is found in mouse pre-implantation embryos after embryonic genome activation. After gastrulation, expression is restricted to germ cells. Limited research has been performed on OCT4 expression in the domestic pig, which is a valuable large animal model in biomedicine. Previously, we generated Oct4-EGFP reporter pigs carrying the genomic sequence of the murine Oct4 gene fused to the EGFP cDNA (Nowak-Imialek et al. 2011 Stem Cells Dev. 20, 1563-1575, 10.1089/scd.2010.0399). In the present study, we used this animal model to analyse the expression profile of the murine Oct4-EGFP transgene in porcine oocytes, in vivo-derived embryos (4-cell embryos, 8- to 16-cell embryos, morulae, and blastocysts) and ovaries. We studied whether the murine Oct4-EGFP transgene mimics the expression pattern of the endogenous OCT4 protein in transgenic pigs. Immature oocytes were isolated from ovaries of Oct4-EGFP transgenic sows (n = 5) using slicing methods. For collection of porcine embryos, wild-type sows were inseminated with sperm from an Oct4-EGFP transgenic boar. Sows were sacrificed 3, 4, and 5 days after insemination, and embryos were recovered by flushing oviducts and uterus and analysed by confocal microscopy. Ovaries obtained from female animals (5–12 months) were enzymatically dissociated and analysed using flow cytometry. Immature oocytes (n = 19) showed a very low, diffuse EGFP signal in cytoplasm. Embryos up to the 4-cell stage (n = 45) did not show Oct4-EGFP transgene expression. For the first time, EGFP fluorescence was detected at the 8-cell stage (n = 29) and a strong EGFP signal was observed in 16-cell stages and morulae (n = 53). In blastocysts from Day 5 (n = 40) EGFP fluorescence was not restricted to the inner cell mass (ICM), but was also seen in the trophectoderm (TE). Expression of EGFP was not detected in ovarian cells (n = 12). Thereafter, we analysed the expression pattern of endogenous OCT4 protein by immunostaining in nontransgenic porcine oocytes and pre-implantation embryos. As in Oct4-EGFP transgenic embryos, no expression of OCT4 was observed in 4-cell embryos (n = 12). Nuclear staining first became visible at the 8-cell stage (n = 12), with a strong signal observed in 16-cell stages and morulae (n = 18). In blastocysts from Day 5 (n = 26), both ICM and TE cell nuclei showed expression of OCT4 protein. These results demonstrate that the Oct4-EGFP transgene expression pattern reproduces the endogenous OCT4 protein expression profile in porcine oocytes and pre-implantation embryos. The Oct4-EGFP transgene was first detected at the 8-cell stage, consistent with embryonic genome activation, which is initiated at the 4-cell stage. However, Oct4-EGFP expression was not detected in ovarian cells. This might be related to the very low expression pattern of the Oct4-EGFP transgene in primary oocytes. In summary, the Oct4-EGFP transgene in the pig provides a useful marker for monitoring pluripotency in pre-implantation embryos after embryonic genome activation. In ongoing experiments, we are analysing the expression profile of the Oct4-EGFP transgene and endogenous OCT4 protein in porcine pre-implantation embryos from Days 8 and 11.

Extracellular matrix structure in SCNT and natural bovine placenta conditions

We have demonstrated previously that retroviral-mediated gene transfer is a promising method to produce transgenic avian, porcine, and bovine embryos. This study was designed to evaluate the development potential of transgenic porcine embryos produced by somatic cell nuclear transfer (SCNT) of fetal fibroblast (pFF) cells transfected by a robust replication-defective retroviral vector harboring enhanced green fluorescent protein (EGFP) or β-galactosidase (LacZ) gene. Moloney murine leukemia virus (MoMLV)-based retroviral vectors encapsidated with VSV-G (vesicular stomatitis virus G) glycoprotein and harboring EGFP or LacZ under the control of β-actin promoter were produced and used to transfect primary pFF cells that were subsequently used for SCNT of enucleated porcine oocytes matured in vitro. Our results showed that all surviving cells after transfection and antibiotic selection expressed the genes without any evidence of replication-competent retrovirus. The fusion, cleavage, and blastocyst rates were 85.6 � 6.5, 53.6 � 6.4, and 12.0 � 5.7% for EGFP; 83.5 � 8.2, 57.5 � 6.3 and 10.1 � 4.1% for LacZ; and 80.5 � 4.2, 60.9 � 8.2 and 12.3 � 4.0% for controls, respectively. Mosaicism was not observed in any of the group as evidenced by the expression of LacZ or EGFP in individual blastomeres of all embryos upon staining with β-galactosidase (for LacZ) or when visualized under UV illumination of an epifluorescent microscope using the fluorescein isothiocyanate (FITC) filter set (for EGFP). Further recloning of EGFP-expressing blastomeres, obtained from 4-cell-stage cloned embryos produced by SCNT of pFF cells infected with EGFP harboring vector, into enucleated metaphase II (MII) oocytes resulted in consistent expression of EGFP in recloned blastocysts. Interspecies SCNT (iSCNT) of transfected pFF into enucleated bovine oocytes could also result in consistent gene expression without any adverse effect on blastocyst rate (5.5 v. 4.9%) compared with non-transfected pFF. These data indicate that the replication-defective retroviral vector used in the present study is robust and independent of the genes inserted. Furthermore, introduction of transgenes by this method does not influence the in vitro development rate of cloned embryos. This work was supported by a grant from Biogreen 21 Program, RDA, Republic of Korea.

As the recipient cytoplast plays a key role in nuclear reprogramming after somatic cell nuclear transfer (SCNT), the aim of this study was to compare the type of cytoplast/karyoplast [metaphase II (MII) oocyte, early zygote, somatic cells] and the chemical (CA) or sperm-mediated/spontaneous activation (SA) on in vitro development of bovine SCNT embryos produced by handmade cloning (HMC). After 17 h of in vitro maturation, a group of cumulus–oocyte complexes (COCs, n = 945) was manually bisected following zona removal and segregated as enucleated (MII hemi-Cyt) or non-enucleated (MII hemi-Kar). Another group of COCs was in vitro-fertilized, and, 4 h after the onset of IVF, zona-free zygotes with 2 polar bodies (n = 490) were manually bisected under fluorescent light to obtain IVF hemi-Cyt and IVF hemi-Kar. A somatic cell (SC) culture from an adult cow was used for HMC procedures (SC Kar). In 5 replications, experimental groups were composed of: zona-intact MII oocytes (parthenote control, PG); zona-intact zygotes (IVF control); MII Cyt + MII Cyt + SC Kar (SCNT control); IVF Cyt + MII Cyt + SC Kar (G1); MII Cyt + IVF Kar (G2); IVF Cyt + IVF Kar (G3); IVF Cyt + IVF Cyt + SC Kar (G4); and MII Cyt + MII Kar (G5). Following reconstruction and electrofusion, groups G1 to G5 were further divided into 2 sub-groups each, 1 being chemically activated (ionomycin/6-DMAP) along with the control groups PG and SCNT, whereas the others were cultured to verify sperm-mediated (G1 to G4) or spontaneous (G5) activation. Embryos were in vitro-cultured in the WOW system for 7 days. Cleavage (Day 2) and blastocyst (Day 7) rates were compared by the chi-square and Fisher tests, respectively. Cleavage rates in G1-SA, G2-SA, and G3-SA were lower than in their CA counterparts, which were similar to controls (Table 1). Such decrease in cleavage in G1-SA and G2-SA may be caused by the manipulation process rather than by sperm-mediation, since the observed rates were very similar to the G5-SA group. Cleavage in G3 and G4 were also similar to controls, most likely due to the fusion of 2 sperm-activated IVF hemi-Cyt. Blastocyst rates were generally higher in CA than in SA sub-groups except for G4, for which SA benefited from 2 sperm-activated cytoplasts. The lower blastocyst yield in SA sub-groups may reflect at least 2 possible mechanisms: an increased level of heteroplasmy (G1 and G2), potentially caused by an insufficient sperm-activated IVF hemi-Cyt or by a blocking effect imposed by the M-phase-derived hemi-Cyt, and/or a disruption in karyokinetic events caused by the manipulation in sperm-activated IVF hemi-Kar (G2 and G3). In G4, both mechanisms were probably attenuated by the use of 2 sperm-activated IVF hemi-Cyt and a SC-kar, analogous to conditions in the SCNT and G5 groups. Table 1.Effect of cytoplast type and activation process on in vitro development of bovine SCNT embryos This study was supported by a grant from CAPES/Brazil.

As animal transgenesis is an essential tool in medicine and agriculture, it is necessary to understand the mechanisms in order to develop novel methods of transgenesis. We intended to determine if the injection of cells or their parts into metaphase II (MII) oocytes after incubating with exogenous DNA can induce transgenesis in embryos. Sperm cells for intracytoplasmic sperm injection (ICSI) in ovine and cumulus cells for NT in bovine were incubated with pCX-EGFP plasmid (5 to 50 ng μL-1) for 5 min in 2.8% Na citrate at 0°C before transfer into a 10% polyvinylpyrrolidone (PVP) droplet and injection into MII oocytes (previously enucleated in NT). In both species, oolemma-ooplasmic vesicles (OOV) of 9 μm diameter obtained from MII oocytes by microsurgery were directly incubated in PVP droplet with same pCX-EGFP concentration. As a control group, pCX-EGFP suspension from PVP droplet was injected into MII oocytes. The NT bovine zygotes were activated in 5 μM ionomycin (Io) for 4 min followed by 1.9 mM DMAP immediately for 3 h. In ICSI ovine, the treatment with DMAP was applied 3 h later. Injected oocytes of OOV and controls were activated as NT in bovine and as ICSI in ovine. Expression of EGFP was determined with fluorescence microscopy under blue light (488 nm) at Days 4 to 7, and data were analyzed by Fisher test (P = 0.05). A group of NT, ICSI, OOV, and control presumptive zygotes were treated with FITC-labeled bovine fragments (100-300 bb) DNA in order to determine the binding sites with exogenous DNA by laser confocal microscope analysis. Quantitative PCR (qPCR) was performed to determine pCX-EGFP copy number at 0, 8, 16, and 24 h after Io in all ovine treatments. Embryos expressing EGDP from all techniques were subjected to FISH with rhodamine-labeled pCX-EGFP plasmid as a probe. In ovine, ICSI and OOV injection green embryos at Day 4 [58% (61/105) v. 21.5% (8/38); P &lt; 0.05] and green blastocysts at Day 7 [71.8% (23/32) v. 66.6% (2/3)] were obtained, respectively. In bovine, green embryos [49.2% (34/69) v. 29.7% (14/47); P &lt; 0.05] and green blastocysts [95.8% (23/24) v. 25.0% (2/8); P &lt; 0.05] were produced by NT and OOV injection, respectively. In controls, no green embryos were obtained in ovine (0/47) and only low rates were observed in bovine [3.0% (2/65)]. Confocal images of zygotes showed specific signal only in cumulus cells, spermatozoa, and OOV The qPCR analysis showed similar plasmid copy number/zygote between treatment and times in ovine (range 30 000-300,000). Embryo FISH images showed 1 to 2 specific signals in ICSI and NT interphases of both species and in OOV ovine metaphases, the latter being direct evidence of transgene integration. These results suggest that the injected cells or cellular parts (OOV) dramatically increase transgenesis in ovine and bovine embryos. Until now, the generation of NT and OOV embryos after short exposure to the DNA construction has not been reported. We are performing embryo transfer and at the moment we have a pregnancy derived from ICSI in ewes. In conclusion, the cellular parts/transgene complex may affect exogenous DNA delivery or its interaction with embryo DNA, facilitating the mechanism of transgenesis in mammals.

In vitro fertilization encounters 2 specific difficulties in the canine species, with no puppies born to date: low penetration rates (10–50%) and high polyspermia (around 50% of fertilized oocytes; Saint-Dizier et al. 2001 J. Reprod. Fert. Suppl. 57, 147–150). The objectives of the study were to test whether intracytoplasmic sperm injection (ICSI), which overcomes these 2 obstacles, could allow production of canine embryos, using in vivo- or in vitro-matured oocytes. The time of ovulation was determined on 8 Beagle bitches from our experimental kennel by blood progesterone assay and transabdominal ultrasound examination. After ovariohysterectomy 82 to 100 h after ovulation, 58 metaphase II (MII) oocytes were collected by tubal flushing. In parallel, 88 oocytes from 6 anoestrus bitches were matured in vitro (M199 + 20% fetal calf serum for 72 h in 5% CO2 at 38°C). Sperm was collected from 1 Beagle dog with excellent fertility record at natural mating. The sperm was diluted 1 : 100 in PBS/BSA without any selection process. Intracytoplasmic sperm injection was performed at 38°C in M199 HEPES + 20% BSA (4-μm injection pipette; 120-μm holding pipette). One motile spermatozoon of normal morphology was injected per oocyte. Injected oocytes were cultured in vitro for 48 h after injection (M199 + 20% fetal calf serum in 5% CO2 at 38°C) in 4-well open dishes. Oocytes were then fixed and DNA and tubulin were stained for observation by confocal microscopy (Chebrout et al. 2012 Microsc. Microanal. 18, 483–492). Among the 58 MII oocytes recovered in vivo, 7.4% lysed at injection and 20% degenerated during the 48 h after injection. Among the 40 injected oocytes still alive, 6 fragmented (15%) and 4 developed as embryos [10%; 2-pronuclei (n = 2), 2-cell and 6-cell). None of the other oocytes showed decondensed female chromatin. Among the 88 oocytes incubated for in vitro maturation, 13 (14.8%) reached MII. These were successfully injected; 48 h after injection, 3 were embryos at the 2-cell stage and 10 were at the MII stage with a condensed sperm head. Fifty-one non-mature oocytes were injected; 31 were at the germinal vesicle (GV) stage and the stage of others was not determined. Of the GV oocytes, 71% degenerated during culture after injection. The 9 surviving oocytes were still at the GV stage with condensed sperm head 48 h after injection. In conclusion, canine embryos can be obtained through ICSI. Nevertheless, this procedure induced low activation rates. Development at later stages, especially after transfer into a recipient female, is to be evaluated, in particular for in vitro-produced MII oocytes, of lower cytoplasmic competence (Viaris et al. 2008 Reprod. Fert. Dev. 20, 626–639).