19 CHANGES IN PLASMA STEROID CONCENTRATIONS DURING GESTATION IN COWS WITH SPONTANEOUS ABORTION OF SOMATIC CELL CLONED FETUSES

2012 ◽  
Vol 24 (1) ◽  
pp. 121
Author(s):  
M. Hirako ◽  
H. Takahashi ◽  
K. Kimura ◽  
N. Adachi ◽  
S. Akagi
Keyword(s):  
Sex Steroids ◽  
Repeated Measures ◽  
Nuclear Transfer ◽  
Plasma Concentrations ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Steroid Synthesis ◽  
Estrone Sulfate

Cloning of mammals by nuclear transfer frequently results in gestational failure with a variety of abnormalities that are likely due to inappropriate epigenetic reprogramming. Monitoring the placental function during gestation is important to clarify the cause of abnormalities in cloned animals. Sex steroids are produced in the bovine placenta and their levels in maternal peripheral blood are a useful measure of placentation. The objective of this study was to investigate changes in plasma concentrations of sex steroids during gestation in cows aborting cloned fetuses. Donor cells for nuclear transfer were obtained from subculture of cumulus cells retrieved from ovarian follicles of a Japanese Black cow. Recipient oocytes were derived from ovaries obtained at an abattoir and matured in vitro. Metaphase II oocytes were enucleated and each fused with a donor cell by DC pulses. Nuclear-transferred oocytes were activated and cultured for 7 days. Embryos developed to the blastocyst stage were each transferred into the uterine horn ipsilateral to the ovary bearing the CL of 39 multiparous Japanese Black and Holstein crossbred cows at 7 to 8 days after the day of standing oestrus (day 0). Fourteen recipient cows were diagnosed pregnant on Day 40 by ultrasonography and 7 cows delivered at full term. The other seven miscarried on Day 66, 81, 85, 89, 97, 104 and 211. Blood was collected from these cows at least once a week following the pregnancy diagnosis. Progesterone, estrone, oestradiol-17β and estrone sulfate in the blood plasma were measured by RIA and were compared with those in pregnant AI cows. Statistical differences at stages of gestation were analysed with repeated-measures ANOVA. In all miscarried cows, progesterone concentrations were similar to those in AI cows until several days before abortion and then rapidly decreased to the basal level. Concentrations of all estrogens stayed low until abortion in six cows aborting by day 104, whereas estrone and oestradiol-17β started to increase around Day 80 and estrone sulfate gradually increased from around Day 50 and started to increase drastically around Day 80 in AI cows. In another cow aborting on Day 211, profiles of estrone and oestradiol-17β were similar to those in AI cows until around Day 150. Thereafter, concentrations of these estrogens gradually decreased to the basal levels by Day 160 and stayed low until abortion. In this cow, gradual increase in estrone sulfate during Day 50 to 80 was not observed, but the difference in the concentration was not statistically significant from AI cows. The following profile of estrone sulfate was similar to those in active estrogens. The fetus was still alive on day 160 and fetal death was confirmed on day 180 by ultrasonography. These results suggest the possibility that developmental or functional failure of placenta associated with steroid synthesis may be a cause of mid-term miscarriage of a cloned fetus.

Preimplantation and postimplantation development of rat embryos cloned with cumulus cells and fibroblasts

Zygote ◽  
2001 ◽  
Vol 9 (2) ◽  
pp. 135-143 ◽  
Author(s):  
Philip Iannaccone ◽  
Greg Taborn ◽  
Ray Garton
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Cell Types ◽  
Blastocyst Stage ◽  
Surrogate Mothers ◽  
Rat Embryos ◽  
Adult Rat ◽  
Rat Fibroblasts

In this report we demonstrate the successful in vitro culture of fertilised embryos from 1-cell to blastocyst stage, albeit in a strain-dependent fashion. We report procedures for the enucleation of rat oocytes; nuclear transfer by injection of nuclei (NT) from adult rat cumulus cells, rat primary embryonic fibroblasts and genetically modified rat fibroblasts; and activation resulting in advanced preimplantation development. Blastocyst stage rat embryos were obtained after in vitro culture of nuclear transfer zygotes at similar frequencies with each of these nuclear donor cell types. Transfer of NT embryos to surrogate mothers leads to implantation of 24% of the zygotes. These results suggest that the nuclei of cultured rat cells, even following genetic modification, can be reprogrammed to support early embryonic development, which is a prerequisite to cloning the rat.

31 FULL-TERM AND LIVE RABBIT CLONES PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 124 ◽  
Author(s):  
F. Du ◽  
J. Xu ◽  
S. Gao ◽  
L. Y. Sung ◽  
D. Stone ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cell Fusion ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Full Term ◽  
Fusion Method ◽  
Nuclear Injection

Transgenic/knockout (KO) rabbits can serve as an excellent animal model for human cardiovascular diseases (CVD) and other diseases. However, the production of transgenic/KO rabbits is hindered by low efficiency of traditional DNA microinjection and the unavailability of embryonic stem cell lines. An alternative approach is to produce transgenic/KO rabbits by somatic cell nuclear transfer (SCNT) using genetically modified somatic cells as nuclear donors. Our initial objective of the study was to prove the feasibility of cloning rabbits by SCNT because rabbit is a difficult species to be cloned. Rabbit oocytes were flushed from the oviducts of superovulated donors treated with the regime of follicle-stimulating hormone (FSH) and human choriani gonadotropin (hCG). Cumulus cells were then denuded from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in M199 + 10% fetal bovine serum (FBS) and confirmed by fluorescence microscopy. Cumulus cells used for nuclear donors were prepared from fresh cumulus-oocytes complexes. The donor nucleus was transferred into a recipient oocyte by either cell fusion or direct nuclear injection method. In the cell fusion method, a small donor cell with the diameter approximately 15–19 µm was transferred into the perivitelline space of an enucleated oocyte; subsequently the somatic cell-cytoplast pair was fused by applying three direct current pulses at 3.2 kV/cm for a duration of 20 µs/pulse. In the direct nuclear injection method, a mechanically lysed donor cell was injected into oocyte cytoplasm with the aid of a piezo-drill system. Fused embryos or injected oocytes were activated by the same electrical stimulation regime described above, and subsequently cultured in M199 + 10% FBS containing 2.0 mM 6-dimethylaminopurine (DMAP) and 5 µg/mL cycloheximide for 2 h. For the in vitro study, cloned embryos were cultured in B2 medium plus 2.5% FBS for 5 days (initiation of activation = day 0) at 38.5°C in 5% CO2 humidified air. For the in vivo study, cloned embryos were cultured for 20–22 h in vitro before transfer into pseudopregnant rabbit recipients. Pregnancy was monitored by palpation and/or ultrasound on Days 14–16 post embryo transfer (ET). The results (Table 1) show that the donor nuclei-introducing rate was higher with nuclear direct injection than with the cell fusion method (P < 0.05). There were no significant differences among subsequent cleavage and development to morula and blastocysts between both methods, although the development rates of cloned embryos via electrically mediated fusion were higher than those derived from the injection group. One recipient in the injection group (1/6, 17%) and six recipients in the fusion group (6/16, 38%) were diagnosed as pregnant. From the fusion group, one full-term but stillborn and one live and healthy clone rabbit were delivered on Days 33 and 31 post-ET, respectively. To our knowledge, this is the second report of full term development of cloned rabbit by somatic nuclear transfer cloning. Our further study is to clone live rabbit offspring with modified transgenic/KO somatic cell lines. Table 1. In vitro development of rabbit cloned embryos with cumulus cells as nuclear donors This work was supported by NIH/NCRR-SBIR grant: 1R43RR020261–11.

5 TRANSCRIPTIONAL PROFILING OF PIG IN IN VIVO, IN VITRO-FERTILIZED, AND NUCLEAR TRANSFER-DERIVED BLASTOCYSTS AND THE DONOR SOMATIC CELL LINE

2008 ◽  
Vol 20 (1) ◽  
pp. 83
Author(s):  
K. M. Whitworth ◽  
L. D. Spate ◽  
R. Li ◽  
A. Rieke ◽  
D. M. Wax ◽  
...  
Keyword(s):  
Cell Line ◽  
Nuclear Transfer ◽  
Transcriptional Profiling ◽  
Reference Sample ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Electrical Activation ◽  
Shock Proteins

The objective of this study was to perform transcriptional profiling between in vivo (IVV), in vitro-fertilized (IVF), and nuclear transfer (NT) blastocyst stage embryos, along with the donor cell line used for NT, in order to identify candidate genes that may contribute to the suboptimal phenotypes of cloned pigs. IVV samples were collected surgically 8 days post-estrus. IVF and NT embryos were transferred into recipient gilts on Day 0 or 1 of estrus and were subsequently collected 6 days later by uterine flush. NT oocytes were activated using one of three methods:NT-1 (electrical activation/fusion), NT-2 (electrical activation/fusion + treatment with proteasomal inhibitor MG 132), or NT-3 (electrical fusion + thimerosal/dithiothreitol (DTT) activation). NT was performed by using pCAG-EGFP positive fetal fibroblast cells to avoid collection of parthenogenetic blastocysts. Donor cells were collected post-NT in pools of 100. Three pools of 10–15 embryos were collected for each treatment. Each pool was analyzed twice, resulting in three biological and two technical replicates. A reference design was used and the reference RNA represented a pool of both reproductive and non-reproductive tissues. Total RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA, USA) and amplified by using an Ovation Ribo-SPIA linear amplification kit (NuGEN Technologies, Inc., San Carlos, CA, USA). Amplified cDNA from blastocysts or cells was labeled with Cy5 and compared to cDNA from the reference sample labeled with Cy3. The cDNAs were hybridized to an in-house printed pig reproductive tissue-specific 19 968 spot cDNA microarray. Microarray images were acquired using a GenePix� 4000B scanner. Spot quality was assessed and results files were constructed using GenePix Pro 4.0. Lowess normalization and analysis was performed in Genespring 7.3.1 (Agilent Technologies, Inc., Palo Alto, CA, USA). Two comparisons were made: IVF versus IVV, and a comparison of all treatments IVV, IVF, NT-1, NT-2, NT-3, and donor cell line. ANOVA (P < 0.05) was performed with the Benjamini and Hochberg False Discovery Rate multiple correction test. The IVF and IVV comparison resulted in 0 differentially detected cDNAs. The IVV, IVF, NT-1, NT-2, NT-3, and donor cell line comparison detected 1477 differentially detected cDNAs, including heat shock proteins (HSPD1 and HSPE1), which are lowly expressed in the donor cell line, and X inactive-specific transcript (XIST), which has higher expression in IVV and IVF compared to that in NT blastocysts. A standard correlation was performed on both comparisons. The R2 value for the IVV and IVF comparison was 0.892, while the R2 value for all samples was 0.716. These results illustrate that IVV and IVF blastocysts, developed within the uterus, are nearly identical. However, a comparison of blastocysts in all treatments including NT and the donor cell line revealed many differentially expressed genes that can be further evaluated for biological function and usefulness as potential markers of quality embryo development after NT.

29 PRODUCTION OF BOVINE CLONED EMBRYOS BY NUCLEAR TRANSFER USING VITRIFIED IMMATURE OOCYTES AS RECIPIENT CYTOPLASTS

2009 ◽  
Vol 21 (1) ◽  
pp. 115
Author(s):  
F. Forell ◽  
C. Feltrin ◽  
L. C. Santos ◽  
A. D. Vieira ◽  
U. M. Costa ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Technological Development ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
National Council ◽  
Immature Oocytes ◽  
Oocyte Recovery

The cryopreservation of immature oocytes is a logistic alternative to make cytoplasts available throughout the year for cloning by somatic cell nuclear transfer (SCNT). Oocyte cryopreservation will help to overcome hurdles related to oocyte availability, seasonality, or sanitary constraints. The objective of this experiment was to determine the efficiency of vitrification of bovine immature oocytes for use as cytoplasts to produce clone embryos. Cumulus–oocyte complexes (COCs) obtained from bovine ovaries by slicing from a local abattoir were selected and vitrified prior to maturation. Vitrification and warming solutions and exposure times were as previously described (Vieira AD et al. 2008 Rep. Dom. Anim. 43, 314–318) with minor modifications. Groups of 15 COCs were loaded in a 5-μL vitrification solution microdrop in beveled-cut straws (0.5 mL), which were plunged into N2L. Following warming, vitrified and control (non-vitrified) oocytes were in vitro-matured for 22 h and 17 h, respectively (Oliveira ATD et al. 2005 Theriogenology 64, 1559–1572). After maturation, cumulus cells were removed and oocytes were selected by the presence of a polar body. Embryo reconstruction by SCNT, carried out by standard micromanipulation procedures using fibroblast cells from adult origin, and in vitro culture to the blastocyst stage (Day 7) were based on our established procedures (Forell F et al. 2008 Acta Sci. Vet. 36, 141–148). Data regarding oocyte recovery following cumulus cell removal, oocyte survival after micromanipulation, and maturation, fusion, cleavage (Day 2), and blastocyst (Day 7) rates were analyzed by the chi-square test. Oocyte recovery (73.0%, n = 558/764 v. 91.4%, n = 529/579), maturation (46.8%, n = 261/558 v. 65.8%, n = 348/529) and cleavage (47.2%, n = 60/127 v. 60.2%, n = 77/128) rates were lower in the vitrified than in the non-vitrified group, respectively (P < 0.05). Conversely, oocyte survival after micromanipulation (77.8% and 78.4%) and fusion (82.1% and 82.3%) and blastocyst (16.7%, 10/60 v. 23.4%, n = 18/77) rates were similar between vitrified and non-vitrified groups. However, the overall efficiency (blastocysts produced from selected COCs) was 3.4-fold lower for vitrified oocytes than controls. In conclusion, the vitrification of immature bovine oocytes was proven as a valuable procedure for the production of blastocysts by SCNT, providing that a strict selection is made following warming, being an alternative resource either for the use of large numbers of oocytes obtained from slaughterhouse ovaries or to overcome seasonal variations in oocyte supply for use in animal cloning. This work was supported by the Brazilian National Council for Scientific and Technological Development (CNPq).

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.

30 IN VITRO EXPRESSION OF BAX AND BCL2 GENES IN NUCLEAR DONOR SKIN FIBROBLAST, CUMULUS, AND GRANULOSA CULTURED CELLS DURING CULTURE AND THEIR SOMATIC CELL NUCLEAR TRANSFER-CLONED EMBRYOS IN INDIAN BUFFALOES (BUBALUS BUBALIS)

2009 ◽  
Vol 21 (1) ◽  
pp. 115
Author(s):  
N. Gupta ◽  
A. Pandey ◽  
S. C. Gupta
Keyword(s):  
Somatic Cell ◽  
Cell Lines ◽  
Granulosa Cells ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Skin Fibroblast ◽  
Cultured Cells ◽  
Donor Cell ◽  
Cumulus Cells

Somatic cell nuclear transfer (SCNT) involves functional changes in the genome which result in low efficiency for the production of viable and cloned embryos. It is primarily due to incomplete reprogramming of genome of donor cell nuclei in the reconstructed embryos (Vassena et al. 2007 Dev. Biol. 304, 75–89). Expression of BCL2 and Bax can be correlated with apoptosis. BCL2 inhibits apoptosis by regulating the release of cytochrome-c and other proteins from mitochondria (Keep et al. 2007 EMBO J. 26, 825–834). Antiapoptotic BCL2 is antiproliferative by facilitating G0. Bax is proapoptotic and accelerates S-phase progression. The dual functions in apoptosis and cell cycle are coordinately regulated by the BCL2 family and suggest that survival is maintained at the expense of proliferation (Zinkel et al. 2006 Cell Death Differ. 13, 1351–1359). The aim of this study was to estimate the relative expression of BCL2 oncogene and Bax gene in regulating apoptosis, in skin fibroblast, cumulus, and granulosa cells in culture, so that ideal-type donor cell lines are developed for higher success rates in SCNT-derived buffalo cloning. The cell lines up to 25th passage were from all the 3 tissue types by previous method (Gupta et al. 2007 Cell Biol. Int. 31, 1257–1264). The cells between passages 5th to 15th were selected as competent donor cells and transferred into enucleated in vitro-matured oocytes from slaughter ovaries. The couplets were activated electrically (1.5 kV cm–2, 15 μs) and chemically (ionomycin, 6-DMAP, CHX, and Cyto-B) and were cultured up to blastocyst. The cDNA were prepared from the growing cells in culture at 5, 10, and 15 passages from all cell lines and SCNT-cloned blastocysts from these cell lines at respective passages for Bax and BCL2 gene expression analysis. Relative expression of these candidate genes was quantified using real-time PCR. The data was analyzed for 1-way ANOVA and post-hoc Duncan multiple range test at P ≤ 0.05 level of significance. The cell proliferation rate in cultured cells at fifth passage was higher in all the 3 cell lines and declined in subsequent passages (range from 1.06 to 0.67). The relative abundance of Bax mRNA in granulosa cell was comparable with skin fibroblasts but significanly higher than cumulus cells at respective passages. BCL2 mRNA expression was significantly upregulated in cumulus cells as compared to granulosa cells but not with skin fibroblasts. The SCNT blastocyst production rates from granulosa were highest (24.28%) as compared to fibroblast (22.6%) and cumulus (21.4%) at passage 10. Level of Bax and BCL2 mRNA in granulosa and fibroblast SCNT blastocysts was not significantly different from IVF (control), whereas cumulus-derived blastocyst showed abnormal patterns with downregulated expression of Bax mRNA and upregulated expression of BCl2 mRNA. Identification of expressed genes in cells and cloned embryos will help to investigate the causes of developmental abnormality due to deregulation of expression of important gene associated with ART.

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

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 55IN VITRO DEVELOPMENT OF ENUCLEATED DOMESTIC CAT OOCYTES RECONSTRUCTED WITH SKIN FIBROBLASTS OF DOMESTIC AND LEOPARD CATS

2004 ◽  
Vol 16 (2) ◽  
pp. 149 ◽  
Author(s):  
C. Lorthongpanich ◽  
C. Laowtammathron ◽  
S. Muenthaisong ◽  
T. Vetchayan ◽  
M. Ketudat-Cairns ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Cell Types ◽  
Blastocyst Stage ◽  
Skin Fibroblasts ◽  
Domestic Cat ◽  
Developmental Potential ◽  
A Cell ◽  
Vitro Development

The domestic cat is a valuable model for studies in assisted reproductive technology in felid species. Therefore, in this experiment we evaluated the in vitro developmental potential of enucleated domestic cat oocytes reconstructed with somatic cells from domestic and leopard cats. Skin fibroblasts were isolated from female domestic and leopard cats. The oocytes were collected by aspiration of follicles from ovaries that were superovulated with 200IU PMSG. In vitro-matured oocytes were enucleated and individual donor cells (diameter 14–16μm) were inserted into the perivitelline space of the enucleated oocyte. Fusion was performed at 26–27h post-maturation by placing a cell-oocyte couplet between both tips of the needle electrode and electrostimulating with a 2-DC pulse (30V, 30μs) in fusion medium containing 0.3M Mannitol+0.1mM MgCl2. Activation was performed 1 to 2h post-fusion by incubation in 7% ethanol at room temperature for 5min followed by cultured in 10μgmL−1 cycloheximide and 1.25μgmL−1 cytochalasin D at 38°C in 5% O2, 5% CO2, 90% N2 conditions. After activation, the reconstructed embryos were cultured in 100-μL droplets of Tyrode’s medium (Gomez et al., 2003 Theriogenology 60, 239–251.) supplemented with 0.3% BSA at 38°C in a 5% O2, 5% CO2, 90% N2 environment for 2d. Then, 8-cell embryos were cultured in 100-μL droplets of Tyrode’s medium supplemented with 10% FCS at 38°C in a 5% O2, 5% CO2, 90% N2environment for 5d. The cleavage rates of oocytes reconstructed with either donor cell types were not different. The percentages of blastocyst formation from parthenogenotes and nuclear transfer embryos derived from domestic cat fibroblasts (8/56, 14.3% and 7/51, 13.7%, respectively) were significantly higher than that for nuclear transfer embryos constructed with leopard cat fibroblasts (3/45, 6.7%). These results indicate that enucleated domestic cat oocytes reconstructed with skin fibroblasts of leopard cats can develop to the blastocyst stage. This experiment was supported by Suranaree University of Technology. Table 1 In vitro development of domestic cat oocytes reconstructed with domestic and leopard skin fibroblasts and parthenogenetic activation

scholarly journals Somatic cell nuclear transfer in horses: effect of oocyte morphology, embryo reconstruction method and donor cell type

Reproduction ◽  
2005 ◽  
Vol 130 (4) ◽  
pp. 559-567 ◽  
Author(s):  
Irina Lagutina ◽  
Giovanna Lazzari ◽  
Roberto Duchi ◽  
Silvia Colleoni ◽  
Nunzia Ponderato ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Reconstruction Method ◽  
Blastocyst Development ◽  
Adult Fibroblasts

The objective of the present work was to investigate and clarify the factors affecting the efficiency of somatic cell nuclear transfer (NT) in the horse, including embryo reconstruction, in vitro culture to the blastocyst stage, embryo transfer, pregnancy monitoring and production of offspring. Matured oocytes, with zona pellucida or after zona removal, were fused to cumulus cells, granulosa cells, and fetal and adult fibroblasts, and fused couplets were cultured in vitro. Blastocyst development to Day 8 varied significantly among donor cells (from 1.3% to 16%, P < 0.05). In total, 137 nuclear transfer-embryos were transferred nonsurgically to 58 recipient mares. Pregnancy rate after transfer of NT-embryos derived from adult fibroblasts from three donor animals was 24.3% (9/37 mares transferred corresponding to 9/101 blastocysts transferred), while only 1/18 (5.6%) of NT-blastocysts derived from one fetal cell line gave rise to a pregnancy (corresponding to 1/33 blastocysts transferred). Overall, seven pregnancies were confirmed at 35 days, and two went to term delivering two live foals. One foal died 40 h after birth of acute septicemia while the other foal was healthy and is currently 2 months old. These results indicate that (a) the zona-free method allows high fusion rate and optimal use of equine oocytes, (b) different donor cell cultures have different abilities to support blastocyst development, (c) blastocyst formation rate does not correlate with pregnancy fate and (d) healthy offspring can be obtained by somatic cell nuclear transfer in the horse.

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