L. N. Moro

Cheetah interspecific SCNT followed by embryo aggregation improves in vitro development but not pluripotent gene expression

The aim of this study was to evaluate the capacity of domestic cat (Dc, Felis silvestris) oocytes to reprogram the nucleus of cheetah (Ch, Acinonyx jubatus) cells by interspecies SCNT (iSCNT), by using embryo aggregation. Dc oocytes were in vitro matured and subjected to zona pellucida free (ZP-free) SCNT or iSCNT, depending on whether the nucleus donor cell was of Dc or Ch respectively. ZP-free reconstructed embryos were then cultured in microwells individually (Dc1X and Ch1X groups) or in couples (Dc2X and Ch2X groups). Embryo aggregation improved in vitro development obtaining 27.4, 47.7, 16.7 and 28.3% of blastocyst rates in the Dc1X, Dc2X, Ch1X and Ch2X groups, respectively (P<0.05). Moreover, aggregation improved the morphological quality of blastocysts from the Dc2X over the Dc1X group. Gene expression analysis revealed that Ch1X and Ch2X blastocysts had significantly lower relative expression of OCT4, CDX2 and NANOG than the Dc1X, Dc2X and IVF control groups. The OCT4, NANOG, SOX2 and CDX2 genes were overexpressed in Dc1X blastocysts, but the relative expression of these four genes decreased in the Dc2X, reaching similar relative levels to those of Dc IVF blastocysts. In conclusion, Ch blastocysts were produced using Dc oocytes, but with lower relative expression of pluripotent and trophoblastic genes, indicating that nuclear reprogramming could be still incomplete. Despite this, embryo aggregation improved the development of Ch and Dc embryos, and normalized Dc gene expression, which suggests that this strategy could improve full-term developmental efficiency of cat and feline iSCNT embryos.

Evaluation of Cheetah and Leopard Spermatozoa Developmental Capability after Interspecific ICSI with Domestic Cat Oocytes

The ICSI procedure is potentially of great value for felids, and it has not been extensively studied in these species. The objectives of this work were to determine the best conditions for ICSI in the domestic cat (DC) to generate interspecific embryos by injecting cheetah (Ch) and leopard (Leo) spermatozoa. Firstly, DC oocytes were matured with insulin-transferrin-selenium (ITS) or without it (MM) and cultured using atmospheric (21%) or low (5%) oxygen tension after ICSI. The group ITS-5%O2 showed the highest blastocyst rate (p < 0.05), 20.9% vs 8.7%, 7% and 6.5%, for MM-21%O2 , MM-5%O2 and ITS-21%O2 , respectively. The best conditions were used to generate the interspecific embryos, together with ionomycin activation (Io) after ICSI. Interspecific embryos resulted in high rates of blastocysts that were not positively affected by Io activation: 32.6% vs 21% for Ch and Ch-Io, 9.8% vs 21% for Leo and Leo-Io, and 20% vs 17.4% for DC and DC-Io. We also evaluated DNA-fragmented nuclei of experiment 1 and 2 blastocysts, using TUNEL assay. The fragmented nucleus proportion was higher in the ITS-5%O2 group, 67.6%. Surprisingly, interspecific blastocysts showed the lowest fragmented nucleus proportion: 27% and 29.9% for Ch and Leo, respectively. We concluded that ITS and 5%O2 improve blastocyst formation in DC, although with a concomitant increase in DNA fragmentation. Most importantly, cheetah and leopard spermatozoa were able to generate blastocysts without artificial activation, which suggests that developmental capacity of wild felid spermatozoa can be evaluated by interspecific ICSI. This technique should be used to assist wild felid reproduction.

Efficient Transgene Expression in IVF and Parthenogenetic Bovine Embryos by Intracytoplasmic Injection of DNA―Liposome Complexes

Transgenic animals constitute an important tool with many biotechnological applications. Although there have been advances in this field, we propose a novel method that may greatly increase the efficiency of transgenic animal production and thereby its application. This new technique consists of intracytoplasmic injection of liposomes, in bovine oocytes and zygotes, to introduce exogenous DNA. In the first experiment, we evaluated embryo development and EGFP expression in In Vitro Fertilization (IVF) embryos injected with different concentrations of exogenous DNA-liposome complexes (0.5, 5, 50, 500 ng pCX-EGFP/μl). The highest EGFP-embryos rates were obtained using 500 ng pCX-EGFP/μl. In the second experiment, we evaluated embryo development and EGFP expression following the injection of DNA-liposome complexes into pre-fertilized oocytes and presumptive zygotes, 16 and 24 h post-fertilization. Approximately 70% of the cleaved embryos and 50% of the blastocysts expressed EGFP, when egfp-liposome was injected 16 h post-fertilization. The percentages of positive embryos for the 24-h post-fertilization and pre-fertilization groups were 30.1 and 6.3, respectively. Blastocysts that developed from injected zygotes were analysed by PCR, confirming the presence of transgene in all embryos. Finally, we examined the embryo development and EGFP expression of parthenogenetic embryos that resulted from the injection of egfp-liposome complexes into pre-activated oocytes, and 3 and 11 h post-activated oocytes. The group with the highest expression rate (48.4%) was the one injected 3 h post-activation. In summary, this study reports the efficient, reproducible and fast production of IVF and parthenogenetic embryos expressing EGFP, by the intracytoplasmic injection of liposomes to introduce the foreign DNA.

Tiger, Bengal and Domestic Cat Embryos Produced by Homospecific and Interspecific Zona-Free Nuclear Transfer

The aim of this study was to evaluate three different cloning strategies in the domestic cat (Felis silvestris) and to use the most efficient to generate wild felid embryos by interspecific cloning (iSCNT) using Bengal (a hybrid formed by the cross of Felis silvestris and Prionailurus bengalensis) and tiger (Panthera tigris) donor cells. In experiment 1, zona-free (ZP-free) cloning resulted in higher fusion and expanded blastocyst rates with respect to zona included cloning techniques that involved fusion or injection of the donor cell. In experiment 2, ZP-free iSCNT and embryo aggregation (2X) were assessed. Division velocity and blastocyst rates were increased by embryo aggregation in the three species. Despite fewer tiger embryos than Bengal and cat embryos reached the blastocyst stage, Tiger 2X group increased the percentage of blastocysts with respect to Tiger 1X group (3.2% vs 12.1%, respectively). Moreover, blastocyst cell number was almost duplicated in aggregated embryos with respect to non-aggregated ones within Bengal and tiger groups (278.3 ± 61.9 vs 516.8 ± 103.6 for Bengal 1X and Bengal 2X groups, respectively; 41 vs 220 ± 60 for Tiger 1X and Tiger 2X groups, respectively). OCT4 analysis also revealed that tiger blastocysts had higher proportion of OCT4-positive cells with respect to Bengal blastocysts and cat intracytoplasmic sperm injection blastocysts. In conclusion, ZP-free cloning has improved the quality of cat embryos with respect to the other cloning techniques evaluated and was successfully applied in iSCNT complemented with embryo aggregation.

Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

DNA fragmentation, transgene expression and embryo development after intracytoplasmic injection of DNA–liposome complexes in IVF bovine zygotes

Summary This study was designed to evaluate the quality and viability of bovine embryos produced by in vitro fertilization (IVF), after intracytoplasmic injection of pCX-EGFP-liposome complexes or pBCKIP2.8-liposome complexes (plasmids that codify the human insulin gene). Cleavage, blastocysts and expanded blastocysts rates of these both groups were not different from that of controls (IVF or IVF embryos injected with liposomes alone; IVF-L). The percentage of EGFP-positive (EGFP+) blastocysts was 41.8%. In Experiment 2, the blastocysts obtained after injection of pCX-EGFP-liposome complexes that did or did not express the transgene, were analyzed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling) assay at days 6, 7 and 8 of culture in vitro(Bd6, Bd7 and Bd8), in order to evaluate DNA fragmentation. The EGFP+ blastocysts showed different proportions of TUNEL-positive cells (T+) at Bd6, Bd7 and Bd8 (91, 73.7 and 99.5%, respectively) while blastocysts without EGFP expression (EGFP-) showed statistically lower numbers of fragmented nuclei (0, 44.6 and 85%, respectively; P < 0.05). There was no evidence of DNA fragmentation in either Bd6 or Bd7 IVF and IVF-L control blastocysts, but T+ nuclei were detected at Bd8 in both groups (66.4 and 85.8% respectively). Finally, IVF blastocysts (n = 21) injected with insulin-liposome complexes, cultured for 6, 7 and 8 days, were transferred to recipient cows. Pregnancy rates of 18.2% (2/11) and 40% (2/5) resulted from the transfer of Bd6 and Bd7 cells, respectively. Two pregnancies developed to term but they were not transgenic for the insulin gene. In conclusion, EGFP expression affects DNA integrity but not embryo development. Moreover, additional transfers are required in order to overcome the drawbacks generated by in vitro culture length and transgene expression.

Bone marrow mesenchymal stem cells as nuclear donors improve viability and health of cloned horses

Introduction Cell plasticity is crucial in cloning to allow an efficient nuclear reprogramming and healthy offspring. Hence, cells with high plasticity, such as multipotent mesenchymal stem cells (MSCs), may be a promising alternative for horse cloning. In this study, we evaluated the use of bone marrow-MSCs (BM-MSCs) as nuclear donors in horse cloning, and we compared the in vitro and in vivo embryo development with respect to fibroblasts. Materials and methods Zona-free nuclear transfer was performed using BM-MSCs (MSC group, n=3432) or adult fibroblasts (AF group, n=4527). Embryos produced by artificial insemination (AI) recovered by uterine flushing and transferred to recipient mares were used as controls (AI group). Results Blastocyst development was higher in the MSC group than in the AF group (18.1% vs 10.9%, respectively; p<0.05). However, pregnancy rates and delivery rates were similar in both cloning groups, although they were lower than in the AI group (pregnancy rates: 17.7% [41/232] for MSC, 12.5% [37/297] for AF and 80.7% [71/88] for AI; delivery rates: 56.8% [21/37], 41.5% [17/41] and 90.1% [64/71], respectively). Remarkably, the gestation length of the AF group was significantly longer than the control (361.7±10.9 vs 333.9±8.7 days), in contrast to the MSC group (340.6±8.89 days). Of the total deliveries, 95.2% (20/21) of the MSC-foals were viable, compared to 52.9% (9/17) of the AF-foals (p<0.05). In addition, the AF-foals had more physiological abnormalities at birth than the MSC-foals; 90.5% (19/21) of the MSC-delivered foals were completely normal and healthy, compared to 35.3% (6/17) in the AF group. The abnormalities included flexural or angular limb deformities, umbilical cord enlargement, placental alterations and signs of syndrome of neonatal maladjustment, which were treated in most cases. Conclusion In summary, we obtained 29 viable cloned foals and found that MSCs are suitable donor cells in horse cloning. Even more, these cells could be more efficiently reprogrammed compared to fibroblasts.

37 Healthy Foals Produced Using Bone Marrow-Mesenchymal Stem Cells as Nuclear Donors in Horse Cloning

Somatic cell nuclear transfer efficiency is based on the capacity of the donor cell to be reset and reprogrammed to an embryonic state. So, the less differentiated the donor cells are, the more easily they could be reprogrammed by a recipient cytoplasm. Failures on appropriate nuclear reprogramming frequently lead to abnormalities associated with the placenta, umbilical cord, birthweight, and limbs. In the present study, we evaluated the efficiency of bone marrow mesenchymal stem cells (BM-MSC) compared with adult fibroblasts (AF) as nuclear donors in horse cloning and evaluated both in vitro and in vivo development of the embryos generated. Moreover, we focused on comparing the health of the foals generated and on the presence of anatomical abnormalities in foals produced from the different treatments. Embryos produced by AI, recovered by uterine flushing, and transferred to recipient mares were used as controls. All variables were analysed by Fisher test (P < 0.05). The cloning procedure was performed according to Olivera et al. (2016 PLoS One 11, e0164049, 10.1371/journal.pone.0164049). Both cleavage and blastocyst rates were higher when MSC were used as nuclear donors (P < 0.05). Cleavage rates were 85.6% (3875/4527) v. 90.2% (3095/3432) and blastocyst rates were 10.9% (492/4527) and 18.1% (622/3432) for AF and MSC groups, respectively. In the AF group, 476 blastocysts were transferred to recipient mares (232 transfers), and in the MSC group, 594 blastocysts were transferred 297 transfers). In the AI control group, 88 embryos were transferred. Pregnancies were diagnosed by transrectal ultrasonography 15 days after embryo transfer in all the groups. Pregnancy rates were similar between both cloning groups (41/232, 17.7% and 37/297, 12.5%for AF and MSC, respectively), but higher in the AI group (71/88, 80.7%). However, significant differences were observed in the birth of viable offsprings among the cloning groups. Despite similar rates of foal delivery (AF, 17/41, 41.5%; MSC, 21/37, 56.7%), a higher proportion of viable foals were obtained from the MSC group (20/37, 54.1%) compared with the AF group (9/41, 22%; P < 0.05). Surprisingly, as in the AI group (63/63, 100%), all of the viable foals obtained using MSC (20/20, 100%) were considered normal and did not show abnormalities associated with cloning. In contrast, in the AF group, only 4/9 (44.4%) were considered normal foals. The defects present in the other 5 foals were related to flexural and angular limb deformities and umbilical cord malformations. These were corrected rapidly with standard treatments or, in the case of the umbilical cords, minor surgery. This study shows for the first time that BM-MSC can be used as nuclear donors in horse cloning and that the foals obtained are as healthy as those produced by AI, showing no abnormalities related to deficiencies in nuclear reprogramming.