scholarly journals 145COMBINED GLUCOSE AND FRUCTOSE SUPPLEMENTATION IN PROTEIN-FREE KSOM IMPROVES PREIMPLANTATION DEVELOPMENT OF BOVINE TRANSGENIC CLONED EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 194 ◽  
Author(s):  
M.M.U. Bhuiyan ◽  
G. Jang ◽  
E.-S. Park ◽  
K.-H. Ko ◽  
H.-Y. Jeon ◽  
...  
Keyword(s):  
Embryo Development ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Total Concentration ◽  
Chemical Activation ◽  
Marker Gene ◽  
Donor Cell ◽  
Preimplantation Development ◽  
Fetal Fibroblasts

The present study investigated the effect of fructose supplementation in protein-free potassium simplex optimization medium (KSOM) on the preimplantation development of bovine transgenic cloned embryos. An expression plasmid containing bovine mutant PrP gene and enhanced green fluorescent protein (eGFP) as a marker gene was constructed and transfected into bovine fetal fibroblasts using FuGene6 (Roche, Indianapolis, IN, USA) as a lipid carrier. Transfected cells were cultured for 5 to 6 days to achieve chromosomal integration of the gene and then used for nuclear transfer. The somatic cell nuclear transfer was carried out by transferring a GFP-expressing donor cell into the perivitelline space of an enucleated oocyte. After electrical fusion and chemical activation, 525 (11 replicates) fused embryos were cultured in KSOM supplemented with 0.01% (w/v) PVA for 192 h at 39°C under 5% CO2, 5% O2 and 90% N2 gas atmosphere. The embryos were randomly allocated for 4 culture groups; KSOM supplemented with 1) 0.2mM glucose, 2) 1.5mM fructose, 3) 0.2mM glucose+1.5mM fructose and 4) vehicle (without glucose and fructose). We used 0.2mM glucose as per formulation of KSOM (Biggers JD et al. 2000 Biol. Reprod. 63, 281–293) and 1.5mM fructose due to its benefical effect on embryo development (Kwun J et al. 2003 Mol. Reprod. Dev. 65, 167–174). As a control experiment, 1043 (17 replicates) in vitro fertilized (IVF) embryos were cultured in the same culture system. The data were analyzed by PROC-GLM using SAS program. In IVF embryos, no significant differences in rates of cleavage (71.7 to 75.5%), morulae (34.1 to 37.1%) and blastocysts formation (21.0 to 24.5%) among the culture groups were observed. In contrast, significantly (P<0.05) higher rate in blastocysts formation (19.2%) was obtained when transgenic cloned embryos were cultured in KSOM supplemented with 0.2mM glucose+1.5mM fructose than with 0.2mM glucose (10.0%). However, the differences in blastocysts formation rates among other culture groups (13.2% for 1.5mM fructose and 11.9% for vehicle) were not significant. Moreover, the rates of cleavage (76.2 to 79.1%), morulae (19.3 to 23.8%) and GFP expression in blastocysts (68.0 to 78.6%) did not differ significantly among the culture groups for transgenic cloned embryos. This study demonstrated that the requirement of energy substrates in culture medium for bovine transgenic cloned and IVF embryos might be different. Now, we are conducting experiments to confirm whether the beneficial effect on transgenic cloned embryo development is due to combined effect of glucose and fructose or due to total concentration of available energy. This study was supported by Biogreen 21-1000520030100000.

scholarly journals Metabolic programming of a Warburg effect-like phenotype in donor fibroblasts prior to somatic cell nuclear transfer

2017 ◽  
Author(s):  
◽  
Bethany Rae Mordhorst
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Nuclear Reprogramming ◽  
In Vitro Development ◽  
Fetal Fibroblasts ◽  
Pharmaceutical Agents ◽  
Vitro Development

Gene edited pigs serve as excellent models for biomedicine and agriculture. Currently, the most efficient way to make a reliably-edited transgenic animal is through somatic cell nuclear transfer (SCNT) also known as cloning. This process involves using cells from a donor (which may have been gene edited) that are typically grown in culture and using their nuclear content to reconstruct a new zygote. To do this, the cell may be placed in the perivitelline space of an enucleated oocyte and activated artificially by a calcium-containing media and electrical pulse waves. While it is remarkable that this process works, it is highly inefficient. In pigs the success of transferred embryos becoming live born piglets is only 1-3%. The creation of more cloned pigs enables further study for the benefit of both A) biomedicine in the development of prognosis and treatments and B) agriculture, whether it be for disease resistance, feed efficiency, gas emissions, etc. Two decades of research has not drastically improved the cloning efficiency of most mammals. One of the main impediments to successful cloning is thought to be due to inefficient nuclear reprogramming and remodeling of the donor cell nucleus. In the following chapters we detail our efforts to improve nuclear reprogramming of porcine fetal fibroblasts by altering the metabolism to be more blastomere-like in nature. We used two methods to alter metabolism 1) pharmaceutical agents and 2) hypoxia. After treating donor cells both methods were used in nuclear transfer. Pharmaceutical agents did not improve in vitro development of gestational survival of clones. Hypoxia did improve in vitro development and we are currently awaiting results of gestation.

Development and spindle formation in rat somatic cell nuclear transfer (SCNT) embryos in vitro using porcine recipient oocytes

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 195-202 ◽  
Author(s):  
Atsushi Sugawara ◽  
Satoshi Sugimura ◽  
Yumi Hoshino ◽  
Eimei Sato
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Spindle Formation ◽  
Fetal Fibroblasts

SummaryCloning that uses somatic cell nuclear transfer (SCNT) technology with gene targeting could be a potential alternative approach to obtain valuable rat models. In the present study, we determined the developmental competence of rat SCNT embryos constructed using murine and porcine oocytes at metaphase II (MII). Further, we assessed the effects of certain factors, such as: (i) the donor cell type (fetal fibroblasts or cumulus cells); and (ii) premature chromosome condensation (PCC) with normal spindle formation, on the developmental competence of rat interspecies SCNT (iSCNT) embryos. iSCNT embryos that had been constructed using porcine oocytes developed to the blastocyst stage, while those embryos made using murine MII oocytes did not. Rat iSCNT embryos constructed with green fluorescent protein (GFP)-expressing fetal fibroblasts injected into porcine oocytes showed considerable PCC with a normal bipolar spindle formation. The total cell number of iSCNT blastocyst derived from GFP-expressing fetal fibroblasts was higher than the number derived from cumulus cells. In addition, these embryos expressed GFP at the blastocyst stage. This paper is the first report to show that rat SCNT embryos constructed using porcine MII oocytes have the potential to develop to the blastocyst stage in vitro. Thus the iSCNT technique, when performed using porcine MII oocytes, could provide a new bioassay system for the evaluatation of the developmental competence of rat somatic cells.

scholarly journals 68EFFECT OF ROSCOVITINE ON FIBROBLASTS' ABILITY TO FORM BLASTOCYSTS AND ESTABLISH PREGNANCIES AFTER BOVINE NUCLEAR TRANSFER

2004 ◽  
Vol 16 (2) ◽  
pp. 155
Author(s):  
A.M. Powell ◽  
P. Graininger ◽  
N. Talbot ◽  
R.J. Wall
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Culture Conditions ◽  
Contact Inhibition ◽  
Serum Starvation ◽  
Blastocyst Development ◽  
Variable Effect ◽  
Return Rates ◽  
Fetal Fibroblasts

Cloning efficiency of fibroblast nuclear transfer is dependent on donor cell chromatin status. Chromatin status is commonly regulated by serum starvation or contact inhibition. We have tested 3 methods of synchronizing chromatin activity, roscovitine exposure (in MEM + 10% serum) for 24h, with serum starvation (0.5% serum) for 5 days or growth to confluence in 10% serum prior to nuclear transfer. Roscovitine, a specific cyclin-dependent kinase (CDK)2 inhibitor, provides a means of precisely synchronizing bovine fetal fibroblasts (BFF) at G0/G1 cell cycle stage. Fibroblasts were from 100-day-old Jersey fetuses. Cells, frozen at passage 2, from fetus 10 are known to produce calves. Fetus 13 cells, frozen at passages 1 and 2, were compared for their ability to serve as nuclear donor cells. Oocytes, either purchased from Bomed or harvested from ovaries obtained from a local slaughterhouse and matured in Ham’s F10, were enucleated between 18–21h post-maturation initiation. Couplets were produced and fused by standard techniques. Embryos were activated 2 to 4 hours after fusion by exposure to ionomycin for 4min and DMAP for 4h. Embryos were then held in CR1aa for 12h before being cultured in G1 media for 3 days and then G2 media for another 3 days (38.5°C and 5% O2 + 5% CO2 + 90% N). On Day 7, good quality blastocysts were transferred to synchronized recipient heifers. The remaining embryos were evaluated after another day in culture. Blastocyst development [(100) X (total blastocysts/fused couplets)] was not influenced by fetus (BFF10, 31±3%; BFF13, 26±2%, P=0.126). However, a higher proportion of blastocysts were produced when fibroblasts were cultured in 0.5% serum (38±3%) compared to culture in 10% serum (29±3%) or in roscovitine (23±2%, P=0.001). Time in culture, as measured by passage, had a variable effect on the fibroblast’s ability to product blastocysts from the three fibroblast culture conditions tested. Passage 1 and 2 fibroblasts responded similarly to the 0.5% and 10% serum treatments (P>0.80). When cultured in roscovitine, passage 1 fibroblasts performed better then passage 2 fibroblasts (29±4% v. 16±3% blastocysts, P=0.010). Embryos have been transferred to 51 recipients to date. Ten recipients have given birth or are still pregnant. The 60-day non-return rate for those animals was 29%, 50%, and 31% for serum starvation, 10% serum, and roscovitine treatments, respectively. BFF10 and BFF13 cells have generated the same non-return rates (33%). In this study, of the 3 methods of synchronizing fibroblast chromatin, serum-starvation had an in vitro advantage. Cells cultured for different lengths of time (passages) responded differently to synchronization treatments. This may reflect a heterogeneous population of cells at early passages. Current non-return rates seem to favor synchronization by contact inhibition. Any advantage roscovitine offers may not be revealed until calving.

33 IN VITRO IMMUNOGENICITY OF SOMATIC CELL NUCLEAR TRANSFER-DERIVED TRANSGENIC CLONED DOGS

2012 ◽  
Vol 24 (1) ◽  
pp. 128
Author(s):  
G. Kim ◽  
H. J. Oh ◽  
J. E. Park ◽  
M. J. Kim ◽  
E. J. Park ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mononuclear Cells ◽  
Donor Cell ◽  
Foreign Gene ◽  
Beagle Dogs ◽  
Fetal Fibroblasts ◽  
Significant Difference

Histocompatible tissue has been generated by somatic cell nuclear transfer (SCNT) and the resultant tissues were not rejected by the immune system of the nucleus donors. In addition, many transgenic animals combined with SCNT have been produced. However, in vitro immunogenicity of transgenic cloned animals originated from the same donor cell with nontransgenic cloned animals has not been assessed until now. The objective of this study was to evaluate the in vitro immunogenicity of cloned dogs with each other, between cloned dogs and transgenic cloned dogs and between transgenic cloned dogs with each other by mixed lymphocyte reaction. In this study, we used cloned beagles (BG1, 2) derived from SCNT using fetal fibroblasts (BF3). Serially, 4 transgenic cloned beagles (Ruppy 1–3, 5) were also genetically engineered from the same donor cell, BF3, with red fluorescent protein (RFP) gene inserted into their genome. We used 2 age-matched healthy female beagle dogs as control dogs. They have different 3 DLA types with all cloned dogs. Peripheral blood mononuclear cells (PBMC) of 2 cloned beagles and 4 transgenic cloned beagles were isolated from whole bloods using Ficoll gradient solution. PBMC from each dog were mixed to auto PBMC, other transgenic cloned dogs and non-related control dogs under the experimental designs. All the mixtures were incubated at 37°C for 4 days, adding BrdU labeling reagent and re-incubated for 24 h. Results are expressed in absorbance mean value ± standard deviation of 450-nm wavelength read by microplate reader. Each cell combination was assayed in 8 replicates. In Experiment 1, PBMC of cloned beagles were combined with equal concentrations of another cloned beagle's PBMC. In Experiment 2, PBMC suspension of Ruppy 1–3, 5 were mixed with equal concentrations of another transgenic cloned beagle's PBMC suspension. In Experiment 3, PBMC suspensions of cloned beagles were mixed with PBMC suspensions of transgenic cloned beagles and reverse reaction was performed. Statistical analysis was performed by using Mann-Whitney U test. In Experiment 1, whereas the absorbance value of mixture of cloned dogs and control dogs shows apparent proliferation, auto mixture of each dog and allo-mixture of BG1 and BG2 show no proliferation (Table 1), indicating immunological factors exposed to PBMC in 2 cloned dogs were compatible. In Experiment 2 among transgenic cloned dogs, no evidence of proliferations in mixed allo-PBMC was shown (Table 1), suggesting in vitro immunogenicity between transgenic cloned dogs was also not shown. In Experiment 3 among cloned dogs and transgenic cloned dogs, no significant difference was found (Table 1). In conclusion, cloned dogs derived from SCNT shared immunological phenotype. Next, immunogenicity among transgenic cloned beagle dogs was not shown despite random insertion of a foreign gene. Lastly, cloned beagles and transgenic cloned beagles show lymphocyte antigen compatibility irrespective of having a foreign gene or not. Table 1.The absorbance values of mixed lymphocytes of 4 transgenic cloned dogs and 2 cloned dogs This study was supported by RNL BIO (#0468-20110001), IPET, MKE (#10033839-2011-13) and Natural Balance Korea.

32 EFFECTS OF FUSION/ACTIVATION METHODS ON DEVELOPMENT OF EMBRYOS PRODUCED BY NUCLEAR TRANSFER OF PORCINE FETAL FIBROBLASTS

2007 ◽  
Vol 19 (1) ◽  
pp. 134
Author(s):  
P. Q. Cong ◽  
E. S. Song ◽  
E. S. Kim ◽  
Z. H. Li ◽  
Y. J. Yi ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Metaphase Plate ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Pulse Number ◽  
Donor Cells ◽  
First Polar Body ◽  
Fetal Fibroblasts

Pigs have become increasingly important in the field of biomedical research, and interest has grown in the use of transgenic cloned pigs as potential xenograft donors. The present study were carried out to investigate the effects of intensity of DC pulse, number of DC pulses, and equilibration before fusion/activation on developmental ability of porcine embryos derived from nuclear transfer. Porcine cumulus-oocyte complexes (COCs) were cultured in modified TCM-199 (mTCM-199) medium for 44 h at 38.5�C, 5% CO2 in air. After in vitro maturation (IVM), metaphase II oocytes were selected for enucleation. Porcine fetal fibroblasts were obtained from a porcine fetus on Day 35 of gestation as donor cells. Oocytes were enucleated by removing, with a micropipette, the first polar body along with adjacent cytoplasm containing the metaphase plate; then a donor cell was injected in contact with the cytoplasm of each oocyte. In experiment 1, several different fusion/activation intensities (two DC pulses of 0.4, 0.8, 1.2, 1.6, and 2.0 kV cm-1 for 30 �s) were carried out to investigate the effect on the development of nuclear transfer embryos. In experiment 2, the reconstructed oocytes were fused and activated with 1, 2, or 3 DC pulses of 1.2 kV cm-1 for 30 �s. In experiment 3, reconstructed oocytes were equilibrated in mTCM-199 medium at 38.5�C, 5% CO2 for 0, 1, 2, 3, 4, 5, and 6 h. After equilibration, the reconstructed oocytes were fused and activated with one DC pulse of 1.2 kV cm-1 for 30 �s in fusion medium. The reconstructed embryos were transferred into PZM-3 medium containing 0.3% BSA for further culture. The rates of embryo cleavage and development of blastocyst stage were evaluated at 48 h and 6-7 days, respectively. The cell numbers of blastocysts were counted by using Hoechst 33342 epifluorescence staining. Data were analyzed by ANOVA and Duncan

In vitro development of green fluorescent protein (GFP) transgenic bovine embryos after nuclear transfer using different cell cycles and passages of fetal fibroblasts

2000 ◽  
Vol 12 (2) ◽  
pp. 1 ◽  
Author(s):  
Sangho Roh ◽  
Hosup Shim ◽  
Woo-suk Hwang ◽  
Jong-taek Yoon
Keyword(s):  
Green Fluorescent Protein ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Blastocyst Formation ◽  
In Vitro Development ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Green Fluorescent ◽  
Vitro Development

Nuclear transfer using transfected donor cells provides an efficient new strategy for the production of transgenic farm animals. The present study assessed in vitro development of nuclear transfer embryos using green fluorescent protein (GFP) gene-transfected bovine fetal fibroblasts. In experiment 1, bovine fetal fibroblasts (BFF) were transfected with linearized pEGFP-N1 by electroporation, and the enucleated oocytes were reconstructed by nuclear transfer of transfected cells (BFF-GFP). The rates of blastocyst formation did not differ significantly between BFF and BFF-GFP (18.2% v. 15.6%). In experiment 2, before nuclear transfer, the donor cell stage was synchronized by serum deprivation or forming a confluent monolayer. The rates of cleavage (67.1% v. 71.8%) and blastocyst formation (15.8% v. 15.5%) did not differ between confluent and serum-starved cells after nuclear transfer. In experiment 3, the effects of different passages of donor fibroblast cells on the development of nuclear transfer embryos were investigated. Donor cells from ‘early’ (at passage 8–16) showed better blastocyst development (18.9%) than those from ‘late’ (at passage 17–32; 10.5%). In conclusion, this study suggests that transgenic somatic cell nuclei from early passages can be reprogrammed more effectively than those from late passages. In addition, GFP, a non-invasive selection marker, can be used to select transgenic nuclear transfer embryos.

Oocyte age and nuclear donor cell type affect the technical efficiency of somatic cloning in rabbits

Zygote ◽  
2003 ◽  
Vol 11 (2) ◽  
pp. 151-158 ◽  
Author(s):  
Rita P. Cervera ◽  
Fernando García-Ximénez
Keyword(s):  
Technical Efficiency ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Cell Types ◽  
Cell Type ◽  
Fetal Fibroblasts ◽  
Somatic Nuclear Transfer ◽  
Metaphase Ii Oocytes

The present study in rabbits compared, in the first experiment, the effect of two commonly used oocyte ages, 13 h and 17 h after ovulation induction treatment, on the technical efficiency of somatic nuclear transfer steps, using fresh cumulus cells as nuclear donors. Recently ovulated metaphase II oocytes (13 h) showed higher fusion (13 h: 83% vs 17 h: 67%, p < 0.05) and in vitro development rates than in vivo slightly aged metaphase II oocytes (morula, 13 h: 74% vs 17 h: 25%, p < 0.05; blastocyst, 13 h: 16% vs 17 h: 8%; p < 0.05). In contrast, activation rate was higher in the 17 h group (13 h: 45% vs 17 h: 67%; p < 0.05). In a second experiment, using recently ovulated oocytes (13 h) as recipients, two donor cell types (from primary cultures of either cumulus cells or fetal fibroblasts) were tested to evaluate their effects on the efficiencies of the different technical steps of somatic nuclear transfer procedure. A better fusion rate was obtained when fetal fibroblasts were used as nuclear donors (cumulus cells: 45% vs fetal fibroblasts: 67%, p < 0.05). No statistically significant differences were detected in cleavage rate regardless of the cell type used (cumulus cells: 44% vs fetal fibroblasts: 60%, p > 0.05). However, in vitro development to morula (cumulus cells: 41% vs fetal fibroblasts: 14%, p < 0.05) and to blastocyst stage (cumulus cells: 27% vs fetal fibroblasts: 3%, p < 0.05) were different between cell types.

scholarly journals Influences of somatic donor cell sex on in vitro and in vivo embryo development following somatic cell nuclear transfer in pigs

2016 ◽  
Vol 30 (4) ◽  
pp. 585-592 ◽  
Author(s):  
Jae-Gyu Yoo ◽  
Byeong-Woo Kim ◽  
Mi-Rung Park ◽  
Deug-Nam Kwon ◽  
Yun-Jung Choi ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell

52 PRELIMINARY DATA ON PIG-BOVINE INTERSPECIES NUCLEAR TRANSFER EMBRYO DEVELOPMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 134 ◽  
Author(s):  
I. Lagutina ◽  
D. Brunetti ◽  
G. Lazzari ◽  
C. Galli
Keyword(s):  
Embryo Development ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Double Pulse ◽  
Cell Stage ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
Fetal Fibroblasts ◽  
Interspecies Nuclear Transfer ◽  
Genome Activation

Interspecies nuclear transfer (NT) is a very important tool for study of nuclear–cytoplasm interactions and somatic cell nucleus reprogramming. We constructed, by means of a zona-free method, NT embryos using bovine (Bo) or porcine (Po) oocytes matured in vitro and bovine fetal fibroblasts (BFF), pig adult fibroblasts (PAF), and pig fetal (PFF) green fluorescent protein (GFP)-positive fibroblasts. Constructs were fused by a double pulse of DC 1.2 kV/cm for 30 µs. At 3–4 h post-fusion, embryos with Bo were activated by 5 µM ionomycin for 4 min and incubated in 2 mM 6-DMAP in SOFaa for 4 h, whereas embryos with Po were activated by a double pulse of DC 1.2 kV/cm for 30 µs in the fusion medium with 1 mM Ca++ and incubated in SOFaa containing 5 µg/mL cytochalasin B in for 4 h. Embryos were cultured in SOFaa in 5% CO2, 5% O2 at 38.5°C. The NT embryo development and GFP expression (D7) were checked. Our results (Table 1) showed that the blastocyst rate of control bovine and pig embryos was 74% and from 20 to 44%, respectively. ‘Pig fibroblasts into Bo’ embryos were arrested at the 8–21-cell stage while ‘BFF into Po’ embryos were arrested at the 4-cell stage. About 84% of ‘PFF GFP+ into Bo’ NT embryos started to express GFP, but only 3.2% (3/95) of the embryos were able to progress through the 16-cell stage suggesting insufficient embryonic genome activation. Overall significantly more ‘Pig fibroblast into Bo’ embryos were able to progress through the 4-cell stage pig developmental block than normal pig NT embryos (57.8 ± 3.5% vs. 47.1 ± 1.3%; t-test, P = 0.02). This study shows that early embryo development is driven by recipient cytoplasm up to the stage when genome activation should occur. The arrest of interspecies NT embryos at the stage of embryonic genome activation suggests that this developmental step is impaired. Table 1. Interspecies NT embryo development This work was funded by grant ISS CS 11 and ESF.

scholarly journals 36COMPARISON OF THE DEVELOPMENTAL POTENTIAL OF CAPRINE NUCLEAR TRANSFER EMBRYOS DERIVED FROM IN VITRO AND IN VIVO MATURED OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 140
Author(s):  
Y. Echelard ◽  
E. Memili ◽  
S.L. Ayres ◽  
M. O'Coin ◽  
L.H. Chen ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Reproductive Tract ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Serum Starvation ◽  
Developmental Potential ◽  
Fetal Fibroblasts ◽  
Student’S T

The objective of this study was to compare the development to the blastocyst stage of reconstructed caprine nuclear transfer (NT) embryos derived from two sources of ova. In vivo oocytes were flushed from the oviduct of superovulated donors by exposing the reproductive tract via a small ventral laparotomy. In vitro oocytes were collected from ovaries supplied by an abattoir located in Purdue, IN. Oocytes were aspirated, cultured in maturation medium (M199 +10% goat serum, 3μgmL−1 LH, 3μgmL−1 FSH and 0.22mM sodium pyruvate), and shipped overnight (38°C, air). Donor cell preparation and NT procedures were as previously reported (Behboodi et al., 2001 Theriogenology 55, 254 abst). Donor cells were transfected female fetal fibroblasts that were synchronized by 4 days of serum starvation, followed by a 10-hour exposure to medium containing 10% FCS. Oocytes were enucleated, karyoplast-cytoplast couplets were reconstructed, fused and then activated simultaneously by a single electrical pulse. Couplets containing in vitro oocytes were incubated in the presence of 5μgmL−1 ionomycin after fusion. Fused couplets were co-cultured in TCM199 with 10% FCS and oviductal epithelial cells for 8–10 days (38°C, 5% CO2). Embryos that developed in vitro to the blastocyst stage were surgically transferred to recipients. Pregnancies were confirmed by ultrasonography. One live kid was delivered on Day 150 of gestation via elective C-section. Southern blotting analysis confirmed that it was derived from the transgenic donor cell line. These experiments show that in vivo matured oocytes not only better support caprine NT embryo development to the blastocyst stage, but also can result in live birth (table). Although fusion and cleavage rates were similar in the two groups, development to the blastocyst stage was significantly higher (Student’s t-test) in the group utilizing in vivo-matured oocytes. In conclusion, this is the first live goat produced from goat NT blastocysts developed in vitro. This suggests that in vivo matured oocytes may be superior to oocytes developed in vitro for generating live animals from NT blastocysts. Table 1

Sign in / Sign up

Export Citation Format

Share Document