Somatic Cell Nuclear Transfer Embryo Production Research Articles

Cytokine supplemented maturation medium improved development to term following somatic cell nuclear transfer (SCNT) in cattle.

In vitro maturation is an important process in the production of embryos. It has been shown that three cytokines, fibroblast growth factor 2, leukemia inhibitory factor and insulin-like growth factor 1 (FLI), increased efficiency of in vitro maturation, somatic cell nuclear transfer (SCNT) blastocyst production, and in vivo development of genetically engineered piglets. Assess effects of FLI on oocyte maturation, quality of oocytes, and embryo development in bovine in vitro fertilisation (IVF) and SCNT. Cytokine supplementation resulted in significant increases in maturation rates and decreased levels of reactive oxygen species. Oocytes matured in FLI had increased blastocyst rates when used in IVF (35.6%vs 27.3%, P <0.05) and SCNT (40.6%vs 25.7%, P <0.05). SCNT blastocysts contained significantly more inner cell mass and trophectodermal cells when compared to the control group. Importantly, SCNT embryos derived from oocytes matured in FLI medium resulted in a four-fold increase in full-term development compared to control medium (23.3%vs 5.3%, P <0.05). Relative mRNA expression analysis of 37 genes associated with embryonic and fetal development revealed one gene had differential transcript abundance in metaphase II oocytes, nine genes at the 8-cell stage, 10 genes at the blastocyst stage in IVF embryos and four genes at the blastocyst stage in SCNT embryos. Cytokine supplementation increased efficiency of in vitro production of IVF and SCNT embryos and in vivo development of SCNT embryos to term. Cytokine supplementation is beneficial to embryo culture systems, which may shed light on requirements of early embryo development.

Cloning by SCNT: Integrating Technical and Biology-Driven Advances.

Somatic cell nuclear transfer (SCNT) into enucleated oocytes initiates nuclear reprogramming of lineage-committed cells to totipotency. Pioneer SCNT work culminated with cloned amphibians from tadpoles, while technical and biology-driven advances led to cloned mammals from adult animals. Cloning technology has been addressing fundamental questions in biology, propagating desired genomes, and contributing to the generation of transgenic animals or patient-specific stem cells. Nonetheless, SCNT remains technically complex and cloning efficiency relatively low. Genome-wide technologies revealed barriers to nuclear reprogramming, such as persistent epigenetic marks of somatic origin and reprogramming resistant regions of the genome. To decipher the rare reprogramming events that are compatible with full-term cloned development, it will likely require technical advances for large-scale production of SCNT embryos alongside extensive profiling by single-cell multi-omics. Altogether, cloning by SCNT remains a versatile technology, while further advances should continuously refresh the excitement of its applications.

The Effects of an In Vitro Oocyte Maturation System and Chlorogenic Acid Supplementation during Embryo Culture on the Development of Porcine Cloned Embryos Derived from Native Vietnamese Ban Pigs

The aim of this study was to improve the production efficiency of Vietnamese native Ban pig embryos using somatic cell nuclear transfer (SCNT). Fibroblast cells from Ban pigs were injected into the enucleated cytoplasts of crossbred gilts, and the reconstructed embryos were subsequently cultured. In the first experiment, cytoplasts were isolated from oocytes matured in either a defined porcine oocyte medium (POM) or in TCM199 medium supplemented with porcine follicular fluid. Both media were supplemented with gonadotropic hormones, either for the first 22 h of in vitro maturation (IVM) or for the entire 44 h of IVM. In the second experiment, the reconstructed SCNT embryos were cultured with or without 50 μM chlorogenic acid (CGA). Furthermore, this study examined parthenogenetic embryos. The IVM medium and duration of hormone treatment did not affect embryo development. CGA supplementation to the culture medium significantly increased blastocyst formation rates in parthenogenetic embryos but not in SCNT embryos. However, CGA supplementation significantly reduced the apoptotic index in blastocysts regardless of embryo source. In conclusion, the IVM method did not affect SCNT embryo production, while CGA supplementation during embryo culture improved the quality of SCNT embryos in indigenous pig breeds.

Oocytes obtained by ovum pick-up from live mares as an alternative to abattoir-derived oocytes for the development of equine embryos produced by somatic cell nuclear transfer

The production of equine embryos by somatic cell nuclear transfer (SCNT) is limited by the availability of immature oocytes. The main source of oocytes for SCNT comes from ovaries obtained from slaughter houses. However, in some countries there is no horse abattoirs or their number is limited which precludes the use of SCNT for the production of equine cloned embryos. Ovum pick-up (OPU) is a reproductive assisted technique which is growing rapidly in the equine industry which allows obtaining oocytes from live mares. OPU is mainly used in combination to ICSI (intracytoplasmic sperm injection) for the production of in vitro embryos. Oocytes collected from live recipient mares by OPU could be an alternative for SCNT in countries in which there are limited number of equine slaughter houses. However, it is unknown the effect of the oocytes’ source on the development of equine cloned embryos. The aim of this study was to compare the development of equine SCNT embryos produced using oocytes either retrieved by OPU or recovered from abattoir-sourced ovaries. For the study, a total of 1128 oocytes were used, of which 663 were from abattoir-sourced ovaries and 495 were from live mares programmed for OPU. The harvested ovaries were transported to the lab within 1 h for processing. The methods used for in vitro maturation (IVM), SCNT andembryo culture in vitro were identical for both sources of oocytes. The rates of maturation, cell fusion, cleavage and blastocyst formation at Day 7 were evaluated. Transferrable grade blastocysts were transferred to recipient mares 4 to 5 days after ovulation and pregnancy was diagnosed at Days 14 and 42 by ultrasound. The maturation rate of OPU-derived oocytes was lower than that of abattoir-derived oocytes (50.3 ± 2.7% vs . 61.9 ± 3.4%; P < 0.05). However, the rates of cell fusion (90.7 ± 2.6% vs. 81.9 ± 5.2%), cleavage (68.8 ± 3.9% vs . 61.9 ± 5.0%) and blastocyst formation (34.3 ± 2.8% vs. 25.4 ± 2.1%) were greater (P < 0.05) for OPU-derived embryos compared with abattoir-derived embryos. While similar proportions of OPU- and abattoir-derived blastocysts initiated pregnancy at Day 14, a greater proportion of OPU-derived blastocysts developed to Day 42 of gestation (13 of 50 embryos) compared with abattoir-derived blastocysts (3 of 27 embryos; P < 0.05). The results show that abattoir-sourced oocytes subjected to IVM have the potential to support the development of SCNT embryos that can initiate pregnancy. However, given the observed difference in subsequent in vivo development, the use of OPU-derived oocytes for equine SCNT embryo production is recommended. Further research is needed to improve the IVM of abattoir-sourced oocytes

A fast and reliable protocol for activation of porcine oocytes

Oocyte activation is physiologically triggered by the sperm during fertilization, however, production of porcine embryos by somatic cell nuclear transfer (SCNT), intracytoplasmic sperm injection (ICSI) or parthenogenetic activation (PA) requires artificial oocyte activation. Although effective protocols for artificial oocyte activation have been developed, current protocols require long exposures to non-specific inhibitors, which do not mimic the physiological process and may have detrimental consequences for embryo development. This study attempted to mimic the physiological activation events induced by fertilization, through the manipulation of Ca2+ and Zn2+ levels, and protein kinase C (PKC) as well as cyclin dependent kinase 1 (CDK1) activities, with the aim of developing an improved protocol for activation of porcine oocytes. In the first experiment, matured oocytes were exposed to ionomycin (Ion) for 5 min, and then treated with a specific CDK1 inhibitor (RO-3306) and/or PKC activator (OAG) for different time intervals. The highest rate of pronuclear (PN) formation (58.8%) was obtained when oocytes were treated with PKCa + CDK1i for 4 h. Second, PN formation and embryo development were evaluated in oocytes exposed for different times to a Zn2+ chelator (TPEN) following Ion treatment. This revealed that 15 min was the minimal exposure time to TPEN required to maximise oocyte activation and embryo development. Next, we observed that treatment with PKCa + CDK1i for 4 h after TPEN for 15 min decreased embryo development compared to TPEN alone. Lastly, we compared the efficiency of the Ion (5 min) plus TPEN (15 min) protocol (IT-20) with a control protocol used in our laboratory (CT-245) for production of PA, SCNT and ICSI embryos. In PA embryos, IT-20 resulted in higher cleavage (72% vs 49.2%) and blastocyst from cleaved embryos (65.5% vs 46.2%) compared to CT-245. In ICSI embryos, higher PN rates were obtained with the IT-20 protocol compared with CT-245 and the non-activated (N-A) group. Moreover, the two protocols were equally efficient for activation of SCNT embryos. Based on these findings, we propose that IT-20 is a fast and effective protocol for activation of porcine oocytes.

Effects of Embryo Aggregation and PXD101 on the In Vitro Development of Mouse Somatic Cell Nuclear Transfer Embryos.

To improve the cloning efficiency of somatic cell nuclear transfer (SCNT) and to establish nuclear transfer embryonic stem cells (NT-ESCs) reliably, it is necessary to produce high-quality blastocysts derived from mice SCNT embryos. Therefore, the present study aims to investigate an optimal method for mouse SCNT embryo production and NT-ESCs derivation by comparing the effects of two methods: the treatment of histone deacetylase inhibitor PXD101 after SCNT, embryo aggregation and their combination treatment. The results suggest that embryo aggregation at four-cell stage and 50 nM PXD101 treated for 10 hours during and after activation could improve both mouse SCNT embryos' development (PXD101: 40.0% vs. 18.5%; p < 0.05; aggregation: 40.2% vs. 18.5%; p < 0.05) and also enhance the isolation rate of NT-ESCs (PXD101: 38.2% vs. 12.5%; p < 0.05; aggregation: 39.0% vs. 12.5%; p < 0.05). The combination of their treatments had a higher development rate (43.6%) and significantly higher NT-ESCs isolation rate (54.7%), therefore, we concluded that the combination of these two methods (50 nM PXD101 treated for 10 hours after SCNT and then aggregated at four-cell stage) is considered as the optimal way for the in vitro development of SCNT embryo and subsequent NT-ESCs isolation in mice, providing a new approach for the practical improvement of mouse cloning techniques and opening new opportunities to improve cloning efficiencies in other species.

Comparative Stepwise Pattern of Reactive Oxygen Species Production during In Vitro Development of Fertilized and Nuclear Transferred Goat Embryos.

BackgroundA unique feature of embryo metabolism is production of reactive oxygen species (ROS). It is well established that during in vitro culture, ROS levels increase over normal ranges observed for embryos developed in vivo. This study evaluates and compares the stepwise pattern of ROS production during in vitro development of reconstructed goat embryos produced by zona-free method of somatic cell nuclear transfer (SCNT). Furthermore, the pattern of ROS production of SCNT embryos were compared with zona free embryos derived from in vitro fertilization (IVF).Materials and MethodsIn this experimental study, zona-free oocytes, SCNT and IVF embryos at different stages of in vitro development (2, 4, 8, 16-cells, morula, and blastocyst) were used for assessment of ROS production using 2, 7-dichloro dihydroflourescein diacetate (DCHFDA) probe and the result were presented as fold increase or decrease relative zona free oocytes.ResultsThe relative level of ROS compared to metaphase-II (MII) oocytes insignificantly decrease during early stages post embryo reconstitution and regained its value by 8-cell and morula stage and, significantly increase compared to MII oocytes by blastocyst stage.ConclusionThe pattern of ROS change in SCNT embryos is similar to zona free IVF derived embryos, except it decrease from two cell stage and regain its value at morula stage. The sudden rise in ROS at blastocyst stage, further emphasizes the special need of IVF and SCNT derived embryos during this stage of development.

Early development of cloned bovine embryos produced from oocytes enucleated by fluorescence metaphase II imaging using a conventional halogen-lamp microscope.

Enucleation of a recipient oocyte is one of the key processes in the procedure of somatic cell nuclear transfer (SCNT). However, especially in bovine species, lipid droplets spreading in the ooplasm hamper identification and enucleation of metaphase II (MII) chromosomes, and thereby the success rate of the cloning remains low. In this study we used a new experimental system that enables fluorescent observation of chromosomes in living oocytes without any damage. We succeeded in visualizing and removing the MII chromosome in matured bovine oocytes. This experimental system consists of injecting fluorescence-labeled antibody conjugates that bind to chromosomes and fluorescent observation using a conventional halogen-lamp microscope. The cleavage rates and blastocyst rates of bovine embryos following in vitro fertilization (IVF) decreased as the concentration of the antibody increased (p<0.05). The enucleation rate of the conventional method (blind enucleation) was 86%, whereas all oocytes injected with the antibody conjugates were enucleated successfully. Fusion rates and developmental rates of SCNT embryos produced with the enucleated oocytes were the same as those of the blind enucleation group (p>0.05). For the production of SCNT embryos, the new system can be used as a reliable predictor of the location of metaphase plates in opaque oocytes, such as those in ruminant animals.

38 ROCKY MOUNTAIN BIGHORN SHEEP (OVIS CANADENSIS CANADENSIS) EMBRYOS PRODUCED USING SOMATIC CELL NUCLEAR TRANSFER

The present objective was to determine if interspecies somatic cell nuclear transfer (SCNT) could be a viable option for producing cloned Rocky Mountain bighorn sheep (RMBS) embryos, a model for other endangered ovine species or sheep of high genetic value. A primary question was whether viable cells from a harvested 5.5-year-old male RMBS (Ovis canadensis canadensis) could be cultured 4 days postmortem for SCNT embryo production. Domestic sheep ovaries from an abattoir were placed in TCM199 holding medium containing Hanks’ salts, kept at ambient temperature, and transported to the laboratory in less than 3 h. Oocytes were collected using 2 methods, aspirating visible follicles (G1) or slicing ovary tissues (G2), and matured in TCM199-based medium. At 20 h postmaturation, oocytes were stripped of cumulus cells and those with an extruded polar body underwent SCNT using ViaGen's protocol. Tissue samples were collected from the RMBS, placed in plastic bags, and shipped to the laboratory for cell culture; that is, tissue disinfection and mincing, placement in cell culture flasks with cell culture medium and into an incubator at 38.5°C in a humidified atmosphere of 5% CO2. At confluency, cells having fibroblast-like cell morphology were cryopreserved using DMSO-containing medium and stored at –196°C. For SCNT, 4-day postmortem donor cells were thawed and cultured for at least 3 days. Matured oocytes were enucleated using a micromanipulation system and Hoechst 33342 to ensure nucleus removal. A donor cell was inserted into an enucleated cytoplast and fusion pulses were applied to reconstruct embryos. Cloned embryos were cultured in modified SOF in 5% CO2, 5% O2, and 90% N2 for 6 days before shipment in a portable incubator at 38.5°C in SOF for analysis on Day 7. Pearson's χ2 test and Fisher's exact test were used to determine statistical differences (α = 0.05). The 60 ovaries yielded 324 oocytes (5.4 oocytes/ovary) and 217 matured (67%) of which 204 were enucleated, 156 fused (77%), and 102 cleaved (65%), producing 25 blastocysts and morulae (25/156 = 16%). G1 resulted in 62 cleaved embryos of 91 fused, whereas G2 resulted in 40 cleaved embryos of 65 fused (χ2 = 0.728; P = 0.393). Using the IETS stage and grading system, 3 blastocysts (one 5–2, one 6–1, one 6–2) and 22 morulae (4–1) appeared viable, but the rest had degenerated. Specifically, G1 produced 3 blastocysts and 8 stage 4–1 embryos versus 14 stage 4–1 embryos in G2 (based on fused or cleaved embryos: χ2 = 2.516, P = 0.113 and χ2 = 3.914, P = 0.048, respectively). Fibroblast-like cell cultures were obtained from 4- and 5-day postmortem tissue samples; however, only Day 4 cultured cells were used for RMBS SCNT embryo production. Although G1 produced more blastocysts than G2, the difference was not statistically significant (P &gt; 0.05).

Scintillating advances on somatic cell nuclear transfer and the implications for human ARTs

Scintillating advances on somatic cell nuclear transfer and the implications for human ARTs

Improved Cloning Efficiency and Developmental Potential in Bovine Somatic Cell Nuclear Transfer with the Oosight Imaging System

In somatic cell nuclear transfer (SCNT) procedures, exquisite enucleation of the recipient oocyte is critical to cloning efficiency. The purpose of this study was to compare the effects of two enucleation systems, Hoechst staining and UV irradiation (hereafter, irradiation group) and Oosight imaging (hereafter, Oosight group), on the in vitro production of bovine SCNT embryos. In the Oosight group, the apoptotic index (2.8 ± 0.5 vs. 7.3 ± 1.2) was lower, and the fusion rate (75.6% vs. 62.9%), cleavage rate (78.0% vs. 63.7%), blastocyst rate (40.2% vs. 29.2%), and total cell number (128.3±4.8 vs. 112.2 ± 7.6) were higher than those in the irradiation group (all p<0.05). The overall efficiency after SCNT was twice as high in the Oosight group as that in the irradiation group (p<0.05). The relative mRNA expression levels of Oct4, Nanog, Interferon-tau, and Dnmt3A were higher and those of Caspase-3 and Hsp70 were lower in the Oosight group compared with the irradiation group (p<0.05). This is the first report to show the positive effect of the Oosight imaging system on molecular gene expression in the SCNT embryo. The Oosight imaging system may become the preferred choice for enucleation because it is less detrimental to the developmental potential of bovine SCNT embryos.

Risk assessment of porcine reproductive and respiratory syndrome virus (PRRSV) transmission via somatic cell nuclear transfer (SCNT) embryo production using oocytes from commercial abattoirs

Somatic cell nuclear transfer (SCNT) technology has become a powerful tool for reproductive biology to preserve and propagate valuable genetics for livestock. Embryo production through SCNT involves enucleation of the oocyte and insertion of a somatic donor cell into the oocyte. These procedures lead to a few small openings on the zona pellucida that may elevate risk of viral infection for the produced SCNT embryos. The oocytes used for SCNT are mainly obtained from abattoirs where viral contamination is almost inevitable. Therefore, a systematic evaluation of risk of disease transmission through SCNT embryo production is necessary prior large scale implementation of this technology in the livestock industry. The objective of the current study was to evaluate the risk of disease transmission via SCNT embryo production and transfer by testing for the presence of porcine reproductive and respiratory syndrome virus (PRRSV) throughout the process of SCNT embryo production. The presence of PRRSV in each step of SCNT embryo production, from donor cells to pre-implantation SCNT embryo culture, was carefully examined using a real-time PCR assay with a sensitivity of five copies per-reaction. All 114 donor cell lines derived from pig skin tissue over a period of 7 years in our facility tested negative for PRRSV. Out of the 68 pooled follicular fluid samples collected from 736 ovaries, only four (5.9%) were positive indicating a small amount of viral molecule present in the oocyte donor population. All 801 Day 7 SCNT embryos produced in four separate trials and over 11,571 washed oocytes obtained in 67 batches over 10 months tested negative. These oocytes were collected from multiple abattoirs processing animals from areas with high density of pig population and correspond to a donor population of over 5828 individuals. These results indicate that the oocytes from abattoirs were free of PRRSV infection and therefore could be safely used for in vitro embryo production. Additionally, the established SCNT embryo production system, including donor cell testing, oocytes decontamination, and pathogen free embryo reconstruction and culturing, bears no risk of PRRSV transmission.

Large scale in vivo risk assessment of bovine viral diarrhea virus (BVDV) transmission through transfer of bovine embryos produced via somatic cell nuclear transfer (SCNT)

The objective was to use the bovine viral diarrhea virus (BVDV) as a model to assess the risk of infectious disease transmission in the system of in vitro embryo production and transfer via somatic cell nuclear transfer (SCNT) technology. The risks of BVDV transmission in the SCNT embryo production were previously evaluated [1]. In that in vitro study, following standard operating procedures (SOP), including pre-nuclear transfer donor cell testing, oocyte decontamination and virus-free cell and embryo culture conditions, SCNT embryos produced were free of detectable viral RNA. The current study focused on the evaluation of the potential risk of disease transmission from SCNT embryos to recipients, and the risk of producing persistently infected animals via SCNT embryo transfer. Blood samples were collected from 553 recipients of SCNT embryos and 438 cloned calves and tested for the presence of BVDV viral RNA via a sensitive real time PCR method. All samples tested were negative. These results, in conjunction with the previous in vitro study, confirmed that the established SCNT embryo production and transfer system is safe and presents no detectable risk of disease transmission.

Comparative Potential of Cultured Skin Fibroblast, Cumulus, and Granulosa Cell to Produce Somatic Cell Nuclear Transfer (SCNT) Preimplantation Embryos in Buffaloes (Bubalus bubalis) in Relation to Gene Expressions

To improve the efficiency of somatic cell nuclear transfer (SCNT)-derived embryos in buffaloes (Bubalus bubalis), skin fibroblast, cumulus, and granulosa cells were cultured up to the 15th passage and cloned embryos were produced from each cell type. At the 15th passage the cumulative population doublings (CPDs) in cumulus cells was higher (60.78) than skin fibroblast (57.12) and granulosa (56.05) cell lines. Gene expression of chromatin remodelling proteins, that is, HDAC1, DNMT1, DNMT3a, and DNMT3b, were comparable at all five passages (P-3, P-6, P-9, P-12, and P-15) groups in cumulus cells but different in skin fibroblast and granulosa cells. Cleavage and blastocyst production rate in cumulus (65.9 and 27.4%)-derived embryos was higher than skin fibroblast (63.8 and 24.3%) and granulosa (62.5 and 22.3%)-derived embryos. Expressions of HDAC1, DNMT1, and DNMT3a mRNA in cumulus-derived blastocysts were similar to IVF blastocysts (control), whereas skin fibroblast and granulosa-derived blastocysts expression was significantly different (p < or = 0.05). DNMT3b mRNA expression in all the three donor cell types and IVF control were similar. The expression pattern of these genes showed the effect of donor cell type with different epigenetic reprogramming capabilities for SCNT embryo production rate. Overall, results indicated that cumulus cells are the best nuclear donor for SCNT.

Risk and prevention of bovine viral diarrhea virus (BVDV) transmission through embryo production via somatic cell nuclear transfer (SCNT) using oocytes from persistently infected donors

The objective was to assess the risk of transmission of bovine viral diarrhea virus (BVDV) through embryo production via somatic cell nuclear transfer (SCNT), with oocytes obtained from persistently infected (PI) donors. Using ultrasound-guided follicular aspiration following superstimulation, oocytes were obtained from five female beef cattle, including three that were PI and two that were negative for BVDV. In the three PI cattle, seven aspirations yielded 32 oocytes (PI-1: three aspirations yielding six oocytes; PI-2: two aspirations yielding 14 oocytes; and PI-3: two aspirations yielding 12 oocytes). The oocyte recovery rate was better in negative control cattle, with 32 oocytes obtained from the two cattle in a single superstimulation and aspiration session. Oocytes were processed individually for SCNT, evaluated, and tested for BVDV. Nearly all (31/32) oocytes from the three PI donors were positive for BVDV by PCR, with detected viral RNA copy number ranging from 1 to 1.1 x 10(5). The proportion of oocytes acceptable for SCNT embryo production (based on oocyte quality and maturation status) was only 16 to 35% from PI donors, but was 81% from control donors. Therefore, routine testing of unacceptable (discarded) oocytes could be an effective approach to identify batches that might contain infected oocytes from PI donors. Identification and removal of high-risk batches of oocytes would minimize the risk of BVDV transmission through SCNT embryo production.

Early metaphase II oocytes treated with dibutyryl cyclic adenosine monophosphate provide suitable recipient cytoplasm for the production of miniature pig somatic cell nuclear transfer embryos

We investigated the effects of in vitro maturation duration and treatment with dibutyryl cyclic adenosine monophosphate (dbcAMP) on the blind enucleation efficiency and developmental competence of miniature pig somatic cell nuclear transfer (SCNT) embryos. Oocytes were cultured for 22 h in NCSU-23 medium with or without 1 mM dbcAMP and then additionally cultured in dbcAMP-free NCSU-23 for 14, 18, or 22 h. Regardless of dbcAMP treatment, the rate of nuclear maturation reached a plateau at 36 and 40 h. However, mitochondrial distribution, a marker for cytoplasmic maturation, differed between the dbcAMP-untreated oocytes at 36 h and dbcAMP-treated oocytes at 40 h. The metaphase II chromosomes were adjacent to the first polar body in 68.8% and 63.5% of the dbcAMP-untreated oocytes at 36 h and dbcAMP-treated oocytes at 40 h, respectively. Furthermore, the blind enucleation efficiency by removing a small volume of cytoplasm was significantly higher in the dbcAMP-untreated oocytes at 36 h (82.9%) and dbcAMP-treated oocytes at 40 h (89.9%) than other groups. The rate of blastocyst formation was highest in the dbcAMP-treated oocytes at 40 h. Hence, this study demonstrated that dbcAMP-treated early metaphase II oocytes are suitable for the production of miniature pig SCNT embryos.

149 RISK ASSESSMENT OF INFECTIOUS DISEASE TRANSMISSION VIA IN VITRO EMBRYO PRODUCTION USING SOMATIC CELL NUCLEAR TRANSFER

Somatic cell nuclear transfer (SCNT) technology is a powerful tool for preservation and propagation of superior genetics of livestock animals. Bovine oocytes derived from abattoirs are usually used in SCNT embryo production. The puncture of the zona pellucida during the nuclear transfer process has raised additional concerns about the risk of disease transmission through application of this new technology. The objective of this study was to use bovine viral diarrhea virus (BVDV) as a model to perform a comprehensive risk assessment on infectious disease transmission in the SCNT system. Thirteen batches of cumulus–oocyte complexes (COC; n = 550) were collected from several abattoirs over 6 months. Two hundred were tested for BVDV before cumulus cell removal. Cumulus cells were removed from the other 350 COC by gentle vortexing in 0.1% hyaluronidase in HEPES-M199 with Hanks’ salts. The cumulus-cell-free oocytes (CFO) were then washed 3 times with FBS-free HEPES-M199 with Hanks’ salts containing penicillin-streptomycin solution (5 μg mL–1). Both COC and CFO were pooled in groups (5/group) and tested for presence of BVDV using sensitive real-time PCR method. Only 2.5% of the COC were BVDV positive and all of the CFO were BVDV negative. Additionally, 293 embryos were produced from 14 different cell lines using the previously described SCNT procedure (Zhou et al. 2008 Mol Reprod Dev. 75, 1281–1289). These embryos were generated using in vitro-matured oocytes collected as 23 different batches over 7 months. The embryos were cultured in vitro to blastocyst stage and then tested for BVDV. All of the 293 SCNT embryos (100%) were BVDV negative. In conclusion, under these SCNT embryo production conditions, a small portion of COC were BVDV positive. However, all CFO and SCNT embryos were BVDV negative. Therefore, the risk of disease transmission using abattoir oocytes and SCNT procedure is relatively low and can be eliminated by using a combination of cumulus cell removal and adequate oocyte washing procedures. F. Arenivas, B. Findeisen, V. Farrar, E. Hwang helped with the SCNT embryo production for this study.

Effect of cytochalasins B and D on the developmental competence of somatic cell nuclear transfer embryos in miniature pigs

In many animals, cytochalasins have generally been used as cytoskeletal inhibitors for the diploid complement retention of somatic cell nuclear transfer (SCNT) embryos. However, limited information is available on the effects of cytochalasins on the in vitro development of SCNT embryos. Hence, we compared the effects of cytochalasin B (CB) and cytochalasin D (CD) on pseudo-polar body (pPB) extrusion, cortical actin filament (F-actin) distribution in porcine parthenogenetic oocytes and in vitro development of SCNT embryos that were reconstructed using foetal fibroblasts in the G0/G1 phase derived from miniature pigs. CB (7.5 microg/ml) and CD (2.5 microg/ml) treatments effectively inhibited pPB extrusion in SCNT embryos. CB (2.5 microg/ml) treatment could not inhibit pPB extrusion and insufficiently destabilized F-actin immediately following artificial activation. In parthenogenetic oocytes treated with 2.5 microg/ml CD, normal reorganization and uniform distribution of cortical F-actin at the cytoplasmic membrane were observed at 8 h after artificial activation; this finding was similar to that of control oocytes. In contrast, parthenogenetic oocytes treated with 7.5 microg/ml CB showed non-uniform distribution of F-actin at 8 h after artificial activation. On day 5 after in vitro cultivation, the blastocyst formation rate of SCNT embryos treated with 2.5 microg/ml CD was significantly higher than that of SCNT embryos treated with 2.5 and 7.5 microg/ml CB (p < 0.05). Hence, the present findings suggest that CD is more effective than CB as the cytoskeletal inhibitor for the production of SCNT embryos in miniature pigs.

Improvement of canine somatic cell nuclear transfer procedure

The purpose of the present study on canine somatic cell nuclear transfer (SCNT) was to evaluate the effects of fusion strength, type of activation, culture media and site of transfer on developmental potential of SCNT embryos. We also examined the potential of enucleated bovine oocytes to serve as cytoplast recipients of canine somatic cells. Firstly, we evaluated the morphological characteristics of in vivo-matured canine oocytes collected by retrograde flushing of the oviducts 72 h after ovulation. Secondly, the effectiveness of three electrical strengths (1.8, 2.3 and 3.3 kV/cm), used twice for 20 micros, on fusion of canine cytoplasts with somatic cells were compared. Then, we compared: (1) chemical versus electrical activation (a) after parthenogenetic activation or (b) after reconstruction of canine oocytes with somatic cells; (2) culture of resulting intergeneric (IG) embryos in either (a) mSOF or (b) TCM-199. The exposure time to 6-DMAP was standardized by using bovine oocytes reconstructed with canine somatic cells. Bovine oocytes were used for SCNT after a 22 h in vitro maturation interval. The fusion rate was significantly higher in the 3.3 kV/cm group than in the 1.8 and 2.3 kV/cm treatment groups. After parthenogenesis or SCNT with chemical activation, 3.4 and 5.8%, respectively, of the embryos developed to the morula stage, as compared to none of the embryos produced using electrical activation. Later developmental stages (8-16 cells) were transferred to the uterine horn of eight recipients, but no pregnancy was detected. However, IG cloned embryos (bovine cytoplast/canine somatic cell) were capable of in vitro blastocyst development. In vitro developmental competence of IG cloned embryos was improved after exposure to 6-DMAP for 4 h as compared to 0, 2 or 6h exposure, although the increase was not significantly different among culture media. In summary, for production of canine SCNT embryos, we recommend fusion at 3.3 kV/cm, chemical activation, culture in mSOF medium and transfer of presumptive zygotes to the oviduct of recipient animals. The feasibility of IG production of cloned canine embryos using bovine cytoplasts as recipient of canine somatic cells was demonstrated.

Development Potential of Transgenic Somatic Cell Nuclear Transfer Embryos According to Various Factors of Donor Cell

The present study was conducted to establish an efficient production system for bovine transgenic somatic cell nuclear transfer (SCNT) embryos, the effect of various conditions of donor cells including cell type, size, and passage number on the developmental competence of transgenic SCNT embryos were examined with their expression rates of a marker gene. An expression plasmid for human prourokinase was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and a human prourokinase target gene into a pcDNA3 plasmid. Three types of bovine somatic cells including two adult cells (cumulus cells and ear fibroblasts) and fetal fibroblasts were prepared and transfected with the expression plasmid using a liposomal transfection reagent, Fugene6, as a carrier. In Experiment 1, three types of bovine cells were transfected at passages 2 to 4, and then trypsinized and GFP-expressing cells were randomly selected and used for SCNT. Developmental competence and rates of GFP expression in bovine transgenic SCNT embryos reconstructed with cumulus cells were significantly higher than those from fetal and ear fibroblasts. In all cell types used, GFP expression rates of SCNT embryos gradually decreased with the progression of embryo development. In Experiment 2, the effect of passage number of cumulus cells in early (2 to 4) and late (8 to 12) passages was investigated. No significant differences in the development of transgenic SCNT embryos were observed, but significantly higher GFP expression was shown in blastocysts reconstructed with cumulus cells at early passage. In Experiment 3, different sizes of GFP-expressing transfected cumulus cells [large (>30 microm) or small cell (<30 microm)] at passages 2 to 4 were used for SCNT. A significant improvement in embryo development and GFP expression was observed when small cumulus cells were used for SCNT. Taken together, these results demonstrate that (1) adult somatic cells as well as fetal cells could serve as donor cells in transgenic SCNT embryo production and cumulus cells with small size at early passage were the optimal cell type, and (2) transgenic SCNT embryos derived from adult somatic cells have embryonic development potential.