34 PIGLETS BORN FROM HANDMADE CLONING

2007 ◽  
Vol 19 (1) ◽  
pp. 135 ◽  
Author(s):  
Y. Du ◽  
Y. Zhang ◽  
J. Li ◽  
P. M. Kragh ◽  
M. Schmidt ◽  
...  
Keyword(s):  
New Technology ◽  
Calf Serum ◽  
Cumulative Effect ◽  
Cumulus Cells ◽  
Widespread Application ◽  
Reconstructed Embryos ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Handmade Cloning

Somatic cell nuclear transfer (SCNT) is probably the most efficient way to produce pigs with targeted genetic modification. Handmade cloning (HMC) is a new technology for SCNT developed recently (Vajta et al. 2005 Reprod. Fertil. Dev. 17, 97–112). However, HMC that resulted in births in cattle was regarded technically difficult in pigs due predominantly to the fragility of MII phase porcine oocytes. The purpose of our present work was to use optimized porcine HMC for production of cloned piglets. Data were analyzed by t-test using SPSS (11.0; SPSS Inc., Chicago, IL, USA). After 42 h in vitro maturation, cumulus cells were removed. Oriented handmade enucleation was performed as described elsewhere (Li et al. concomitant abstract). Briefly, oocytes that were partially digested with 3.3 mg mL-1 of pronase for 20 s were removed of polar bodies (PB) and adjacent small volume of cytoplasm by manual bisection in HEPES-buffered TCM-199 medium supplemented with 2% calf serum and 2.5 �g mL-1 of cytochalasin B. Halves without PB were collected as putative cytoplasts. In vitro-cultured porcine fetal fibroblasts were used as donor cells. After cytoplast-fibroblast pairing, fusion and activation of fused cytoplast-fibroblast pairs (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Clon Stem Cells 7, 199–205), reconstructed embryos were cultured in a modified well of the well (WOW) culture system (Feltrin et al. 2006 Reprod. Fertil. Dev. 18, 126 abst) with porcine zygote medium-3 (PZM3) supplemented with 4 mg mL-1 of BSA. The cumulative effect of the optimization steps has resulted in considerably improved in vitro efficiency, shown as 64 � 2.3 (mean � SEM) reconstructed embryos from 151.3 � 4.8 oocytes could be obtained per day after 3–4 h manual work, including a 1-h pause between fusion and activation. One-half (50.1 � 2.8%) of the reconstructed embryos developed to the blastocyst stage after 7 days, a rate that was significantly higher than that obtained with traditional cloning (TC; 27.7 � 2.2%; P < 0.01). To compare the transfer efficiency between HMC and TC, blastocysts from both HMC and TC produced by using nuclear donor cells of different origin, respectively, to identify the offspring were transferred surgically to synchronized recipients. Of 6 pregnancies produced, 2 are ongoing, 2 were lost, and 2 term litters of 3 and 10 piglets were born. Live birth/transferred embryo efficiencies for HMC and TC are 17% (10/58) and 15% (3/20). According to our knowledge, a litter size of 10 cloned healthy piglets, as achieved in this study, is the highest one that ever has been reported. Our data suggest that porcine HMC is a very promising method for SCNT and may promote its widespread application for various purposes.

56 TRANSGENESIS BY HANDMADE CLONING USING EGFP-TRANSFECTED YUCATAN FIBROBLASTS

2007 ◽  
Vol 19 (1) ◽  
pp. 146
Author(s):  
P. M. Kragh ◽  
Y. Du ◽  
J. Li ◽  
Y. Zhang ◽  
L. Bolund ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Polar Body ◽  
Cumulus Cells ◽  
Miniature Pig ◽  
Egfp Gene ◽  
Fusion Procedure ◽  
Reconstructed Embryos ◽  
Fetal Fibroblasts ◽  
Handmade Cloning

Somatic cell nuclear transfer (SCNT) offers the possibility of pig transgenesis. Importantly, genetic manipulations can be performed in cells isolated from special breeds followed by SCNT into enucleated oocytes isolated from slaughterhouse ovaries. In the present study, we established production of Yucatan blastocysts by the handmade cloning (HMC) technique using non-transgenic fibroblasts from the Yucatan miniature pig, and produced transgenic blastocysts using enhanced green fluorescent protein (EGFP)-positive Yucatan fetal fibroblasts. For transgenesis, Yucatan fibroblasts from a 40-day old fetus were transfected with a vector containing an EGFP gene and a neomycin-resistance selection gene by lipofection. Well separated neomycin-resistant colonies were isolated, expanded, and used for HMC. For HMC, cumulus–oocyte complexes were aspirated from ovaries of slaughterhouse sows and matured for 41 h. Subsequently, the cumulus cells were removed in hyaluronidase, and zonae pellucidae were partially digested by incubation in pronase. Oocytes with a visible polar body (PB) were subjected to oriented bisection. Less than half of the cytoplasm adjacent to the PB was removed with a microblade. The remaining parts, i.e. cytoplasts, were used as recipients for embryo reconstruction. Reconstructed embryos were produced by a two-step fusion procedure. At the first step, one cytoplast was fused with one fibroblast in the absence of Ca2+. After one h, the cytoplast-fibroblast pair and another cytoplast were fused and activated simultaneously in the presence of Ca2+, and subsequently cultured in cytochalasin B and cycloheximide for 4 h. The development of reconstructed embryos to the blastocyst stage was determined after 7 days of in vitro culture. When using non-transgenic and EGFP-positive Yucatan fetal fibroblasts, the rate of blastocyst formation (mean � SEM) were 36 � 7% (36/102) and 42 � 7% (32/77), respectively. In conclusion, the HMC technique was very efficient for production of blastocysts of Yucatan miniature pig origin using both non-transgenic and EGFP-positive fetal fibroblasts.

65 CLONED EMBRYOS FROM HORSE SOMATIC CELLS AND ENUCLEATED BOVINE AND OVINE OOCYTES AND THEIR DEVELOPMENTAL COMPETENCE

2008 ◽  
Vol 20 (1) ◽  
pp. 113
Author(s):  
H. M. Zhou ◽  
B. S. Li ◽  
L. J. Zhang
Keyword(s):  
Somatic Cell ◽  
Somatic Cells ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Electrical Pulses ◽  
Mongolian Horse

The objective of this study was to investigate the reprogramming potential of equine somatic cell donor nuclei in either bovine or ovine recipient oocyte cytoplasmic environments. Heterogeneous embryos were reconstructed by somatic cell nuclear transfer (NT). The percentage of fusion and developmental competence, assessed by rates of cleavage and morula and blastocyst formation, were determined. Skin fibroblast cells, obtained from the ear of an adult female Mongolian horse, were dissociated using 0.25% trypsin and cultured in vitro in a humidified atmosphere of 5% CO2 in air at 37°C. Donor somatic cells were serum-starved before NT and used between passages 4 and 6. Bovine and ovine oocytes derived from slaughterhouse ovaries were matured in vitro for 17–19 and 22–24 h, respectively, in a humidified atmosphere of 5% CO2 in air at 38.5°C, before they were enucleated and used as recipient cytoplasts. The fibroblasts were injected under the zona pellucida of the cytoplasts and electrically fused by 2 DC electrical pulses of 1.58 kV cm–1 for 10 μs, with an interval of 0.13 s. The reconstructed embryos were then activated with 5 μm ionomycin in H-M199 for 5 min and then in 2 mm 6-DMAP for 4 h. The equine-bovine and equine-ovine reconstructed embryos were co-cultured, respectively, with bovine and ovine cumulus cells in synthetic oviduct fluid supplemented with amino acids (SOFaa) and 10% fetal calf serum (FCS) for 168 h. The data were analyzed with ANOVA and differences among the groups were evaluated with t-test. The results of the percentages of fusion, cleavage, and development to morula (8 to 64 cells) and blastocyst stages of equine-bovine and equine-ovine heterogeneous embryos are shown in Table 1. This study demonstrates that heterogeneous embryos can undergo early embryonic divisions and that reprogramming of equine fibroblast nuclei can be initiated in foreign cytoplasts. It appears that embryos reconstructed with equine somatic nuclei and ovine cytoplasts have a higher developmental potential than those using bovine cytoplasts. Table 1. Developmental competence of equine-bovine and equine-ovine reconstructed embryos

scholarly journals 28 CLONED EMBRYOS CAN BE PRODUCED USING DONOR CELLS OBTAINED FROM A 72-HOUR COOLED CARCASS

2005 ◽  
Vol 17 (2) ◽  
pp. 164 ◽  
Author(s):  
S. Arat ◽  
H. Bagis ◽  
H. Odaman Mercan ◽  
A. Dinnyes
Keyword(s):  
Calf Serum ◽  
Calcium Ionophore ◽  
Cumulus Cells ◽  
Penicillin G ◽  
Developmental Potential ◽  
Viable Cells ◽  
Donor Cells ◽  
A Cell ◽  
Vitro Development

There are few reports on the use of cells from a dead mammal for nuclear transfer (NT). So far, most calves have been cloned from live adult cows or fresh fetal samples. The ability to produce cloned animals using postmortem tissue can provide an additional application to the field of NT. This study was conducted to investigate whether viable cells could be obtained from tissues chilled for 72 h and whether these cells could be used for NT. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM199 supplemented with 10% fetal calf serum (FBS), 50 μg/mL sodium pyruvate, 1% v:v penicillin-streptomycin (10,000 U/mL penicillin G, 10,000 μg/mL streptomycin), 10 ng/mL EGF, 0.5 μg/mL FSH, and 5 μg/mL LH. A cell line (MC) was established from leg muscle of a cow carcass stored at 0°C for 72 h. Tissues from muscle were cut into small pieces. Tissue explants were cultured in DMEM-F12 supplemented with 10% FBS at 37°C in 5% CO2 in air. Bovine granulosa cells (GC) were isolated from ovarian follicles and used for NT as control cells. Prior to NT, all somatic cells were allowed to grow to confluency (G1/G0) in DMEM-F12 medium supplemented with 10% FBS. Cumulus cells were removed by vortexing with hyaluronidase at 18 h after the start of maturation. Matured oocytes labeled with DNA fluorochrome Hoechst 33342 were enucleated under UV to ensure full removal of the chromatin. A single cell was inserted into the perivitelline space of the enucleated oocyte. Oocyte-cell couples were fused by a DC pulse of 133V/500 μm for 25 μs. After fusion, NT units were activated using a combination of calcium ionophore (5 μM), cytochalasin D (2.5 μg/mL) and cycloheximide (10 μg/mL) and cultured for 7 days in BARC or G1.3-G2.3 medium. Differences (developmental potential and cell numbers) among groups were analyzed by one-way ANOVA after arcsin square transformation. The results are summarized in Table 1. The results suggest that viable cells can be obtained from muscle of a cow carcass stored at cold temperature for 72 h and that these cells have ability to generate NT blastocysts at rates similar to those obtained with fresh GCs. In addition, G1.3 and G2.3 culture medium supported embryo development better than BARC medium. Table 1. In vitro development of NT embryos This study was supported by a grant from TUBITAK, Turkey (VHAG-1908 and Turkey-Hungary bilateral project VHAG-2022).

scholarly journals 75 RABBIT NUCLEAR TRANSFER WITH CULTURED SOMATIC CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 187 ◽  
Author(s):  
F. Yang ◽  
B. Kessler ◽  
S. Ewerling ◽  
E. Wolf ◽  
V. Zakhartchenko
Keyword(s):  
Nuclear Transfer ◽  
Cultured Cells ◽  
Somatic Cells ◽  
Cumulus Cells ◽  
Cell Stage ◽  
Electric Pulses ◽  
Donor Cells ◽  
Fetal Fibroblasts

Cloned rabbits have been obtained by somatic cell nuclear transfer (SCNT) only with fresh, non-cultured cumulus cells (Chesne et al. 2002 Nat. Biotechnol. 20, 366–369). For the purpose of generating transgenic animals by SCNT, donor cells must be cultured and modified prior to use as nuclear donors. The objective of this study was to optimize the SCNT procedure using cultured cumulus or fibroblast cells. MII oocytes were harvested from superovulated Zika rabbits, and maternal chromosomes were removed by demecolcine-assisted enucleation (Yin et al. 2002 Biol. Reprod. 67, 442–446). Two types of somatic cells originating from Ali/Bass rabbits were used as nuclear donors: cumulus cells collected from in vivo-matured oocytes and cultured for 1–5 passages, and primary fetal fibroblasts obtained from Day 16 fetuses and grown to confluence or starved for 4–5 days. Somatic donor cells and recipient cytoplasts were fused with 2 electric pulses (1.95 kV/cm, 25 μs each, 1 s interval). Twenty to 40 min after fusion, cloned embryos were activated first with the same electropulses as for fusion, and then immediately followed by 1 h incubation in 2 mM 6-dimethylaminopurine and 5 μg/mL cytochalasin B in culture medium (B2 medium supplemented with 10% FCS). Cloned embryos were either transferred at the 2- and 4-cell stage to asynchronized recipients or cultured in vitro for 6 days. Data were compared using chi-square test, and differences were considered significant when P < 0.05. Our results demonstrate that cloned rabbits can be produced by SCNT with cultured cells but the efficiency of this technique is still very low irrespective of the type of donor cells. Table 1. Development of cloned embryos derived from somatic cells This research was supported by the Therapeutic Human Polyclonals, Inc.

scholarly journals 75HIGHLY EFFICIENT AND RELIABLE CHEMICALLY ASSISTED ENUCLEATION METHOD FOR HANDMADE CLONING IN CATTLE AND SWINE

2004 ◽  
Vol 16 (2) ◽  
pp. 159 ◽  
Author(s):  
G. Vajta ◽  
T.T. Peura ◽  
L. Lai ◽  
C.N. Murphy ◽  
R.S. Prather ◽  
...  
Keyword(s):  
Uv Light ◽  
Polar Body ◽  
Cumulus Cells ◽  
Dependent Manner ◽  
Uv Illumination ◽  
Reconstructed Embryo ◽  
Fetal Fibroblasts ◽  
Membrane Protrusions ◽  
Handmade Cloning

In bovine and porcine nuclear transfer, most traditional enucleation procedures require potentially harmful chromatin staining and UV illumination. The purpose of our work was to find an efficient and reliable chemically-assisted procedure for enucleation connected to the handmade cloning (HMC) technique without chromatin staining. Slaughterhouse-derived oocytes were collected and matured in vitro. At 21 (bovine) or 43 (porcine) h after the start of maturation, cumulus cells were removed with vortexing and oocytes were further incubated in the maturation medium supplemented with 0.5μgmL−1 demecolcine for 2h. Subsequently, zonae pellucidae were digested with 2mgmL−1 pronase in the presence of 10% cattle serum (CS) for 6 to 8min and washed in HEPES-buffered TCM-199 medium and 20% CS. Bisection was performed in the same medium by hand under a stereomicroscope by using a microblade. A small membrane protrusion observable on the surface of oocytes was used as an orientation point. One-third of the cytoplasm connected to this protrusion was removed, and the cytoplasts and karyoplasts were collected separately. Bovine cytoplasts were used as recipients for HMC experiments (Vajta et al., 2003, Biol. Reprod. 68, 571–578) with fetal fibroblasts as donors, and reconstructed embryos were cultured for 7 days. In Experiment 1 (3 replicates), the possibility of oriented bisection at different time points was determined on a total of 225 bovine oocytes. At 5, 15, 25, 35 and 55min after the end of pronase digestion 64, 91, 93, 72 and 59% of oocytes had membrane protrusions (P&lt;0.05 between all groups, SAS Genmod) illustrating the time-dependent manner of the protrusion. In Experiment 2, the efficiency and reliability of enucleation was measured. Bisection was performed between 5 and 35min after pronase digestion. Subsequently both supposed cytoplasts and karyoplasts were stained with Hoechst and investigated under UV light. In cattle (9 replicates), bisection was successfully performed in 94% (519/552) of oocytes, and 98% (507/517) of those bisected were enucleated, i.e. the chromatin was entirely in the presumptive karyoplast. In swine (3 replicates), 91% (302/331) of oocytes were successfully bisected and 95% (280/296) were enucleated. In Experiment 3 (cattle; 4 replicates), blastocyst per reconstructed embryo rates were 47% (139/293), illustrating the high developmental ability in vitro. Considering that no oocyte selection based on the presence of polar body was performed, the above system seems to be more efficient and reliable than other enucleation methods. Moreover, expensive equipment (inverted fluorescent microscope) and a potentially harmful step (staining and UV illumination) can be eliminated from the HMC without compromising the high in vitro efficiency.

scholarly journals 28 SIMPLIFIED ACTIVATION METHOD TO IMPROVE THE IN VITRO DEVELOPMENT OF HANDMADE CLONED (HMC) PORCINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 114
Author(s):  
Y. Du ◽  
Z. Yang ◽  
B. Lv ◽  
L. Lin ◽  
P. M. Kragh ◽  
...  
Keyword(s):  
Cell Number ◽  
Activation Method ◽  
The Other ◽  
Electrical Activation ◽  
In Vitro Development ◽  
Reconstructed Embryos ◽  
Fetal Fibroblasts ◽  
Group 2 ◽  
Vitro Development

Delayed activation is commonly used in pig somatic cell nuclear transfer (SCNT) where electrical activation is followed by chemical activation. However, chemical incubation of several hours (up to 4 or 6) is logistically not very convenient even though handmade cloning (HMC) could improve the overall efficiency of pig cloning (Du et al. 2007 Theriogenology 68, 1104–1110). It was reported that a brief exposure of cycloheximide (CX) before electrical activation could significantly increase developmental rate and total blastocyst cell number when simultaneous activation was performed in micromanipulator-based pig cloning (Naruse et al. 2007 Theriogenology 68, 709–716). The purpose of our present work is to investigate whether such activation method is also applicable for pig HMC. Data were analyzed by t-test using SPSS (11.0, SPSS Inc., Chicago, IL, USA). After 42 h in vitro maturation, cumulus cells were removed. In vitro-cultured porcine fetal fibroblasts were used as donor cells. Cytoplast-fibroblast pairing, electrical fusion and activation of fused cytoplast-fibroblast pairs were performed as described previously (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Cloning Stem Cells 7, 199–205). Three groups were compared due to different activation protocol. In Group 1 (control), reconstructed embryos were cultured in porcine zygote medium 3 (PZM3) supplemented with 4 mg mL–1 BSA, 5 μg mL–1 cytochalasin B (CB), and 10 μg mL–1 CX for 4 h. In Group 2 (CX priming), fused pairs and the other halves of cytoplasts were incubated in HEPES-buffered TCM-199 medium supplemented with 10% calf serum, 10 μg mL–1 CX for 10 min just before the second fusion or electrical activation. In Group 3 (CB + CX priming), treatment similar to Group 2 was performed except that additional 5 μg mL–1 CB was added for the 10-min incubation. Reconstructed embryos were in vitro cultured in the well of the well (WOW) system for 6 days. Blastocyst rates and total cell numbers of Day 6 blastocysts were evaluated. As illustrated in Table 1, embryos pretreated with both CB and CX gave the best results, with better blastocyst formation (53.8 ± 4.8%; mean ± SEM) and higher cell number (77.2 ± 5.4) compared to the other 2 groups. Our data suggested that CX and CB priming could be used as a solution to the long chemical incubation in porcine SCNT by HMC, making the embryos more receptive to electrical activation. Table 1.In vitro development of HMC reconstructed embryos with different activation protocols

scholarly journals 63 IMPROVED IN VITRO DEVELOPMENT OF PORCINE EMBRYOS PRODUCED BY NUCLEAR TRANSFER, IVF AND PARTHENOGENESIS

2005 ◽  
Vol 17 (2) ◽  
pp. 181 ◽  
Author(s):  
D. Sage ◽  
P. Hassel ◽  
B. Petersen ◽  
W. Mysegades ◽  
P. Westermann ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Culture Media ◽  
Cumulus Cells ◽  
Cell Number ◽  
Foster Mother ◽  
Chi Square ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate ◽  
Parthenogenetic Embryos

Porcine nuclear transfer (NT) is an inefficient process and it is necessary to use as many as 120 NT embryos for each foster mother to obtain small litters of live piglets. In these experiments, we evaluated the effects of culture atmosphere and medium on the development of NT embryos by monitoring blastocyst rate and cell number of Day 6 blastocysts. Age matched IVF and parthenogenetic embryos were also evaluated for comparison. For all experiments a pool of oocytes was aspirated from ovaries collected in a local abattoir. Following aspiration, oocytes were allowed to mature for 40 h in North Carolina State University (NCSU)-37 medium (supplemented with cAMP and hCG/eCG for the first 22 h). After removal of the cumulus cells, denuded oocytes with polar bodies were selected for NT, enucleated, fused with fetal fibroblasts, and sequentially activated electrically and chemically by 3 h of treatment with 6-dimethylaminopurine (6-DMAP). A second group of oocytes from the same denuded pool were maintained in TL-HEPES medium and activated in parallel with the NT group to produce parthenogenetic embryos. A third group was fertilized with frozen-thawed epididymal semen and co-cultured for ∼12 h to give IVF embryos. All three treatment groups were subdivided into a control subgroup and an experimental subgroup. In the first experiment, we compared the effects of atmosphere (20% vs. 5% oxygen) on in vitro embryonic development in NCSU-23 medium. In the second experiment, we used only the 5% oxygen concentration and compared different culture media. One subgroup was maintained in standard NCSU-23 medium and the second subgroup was cultured in a two-step system for the first 58 h in modified NCSU-23 (without glucose but supplemented with 2.0 mM lactate and 0.2 mM pyruvate), followed by addition of glucose to give a final concentration of 5.55 mM. Data were statistically analyzed by analysis of variance and chi square test. Blastocyst rate and mean cell number in all three embryo groups were improved under 5% oxygen. The most dramatic effect was observed in the NT group, in which the blastocyst rate increased significantly (P < 0.001) from 6.7% ± 5.9 (n = 279) to 19.6% ± 8.9 (n = 250) and mean cell number increased from 17.7 ± 12.1 to 25.8 ± 10.3 cells per blastocyst. With 5% oxygen there was also an increase of blastocyst rates and mean cell numbers in both IVF and parthenogenetic groups. In the second experiment, blastocyst rate for NT embryos increased significantly (P < 0.05) from 21.8% ± 7.6 (n = 242) in conventional NCSU-23 to 31.5% ± 11.0 (n = 271) in the modified system whereas there was almost no difference in the mean cell number of both groups (29.2 ± 4.3 vs. 31.5 ± 5.3). In the groups of IVF and parthenogenetic embryos no difference was found. These results indicate that both the reduced oxygen and the modified culture medium are important for pre-implantation development of porcine nuclear transfer embryos.

102 RABBIT CLONING: HISTONE ACETYLATION STATUS OF DONOR CELLS AND CLONED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 168
Author(s):  
V. Zakhartchenko ◽  
F. Yang ◽  
R. Hao ◽  
E. Wolf
Keyword(s):  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Epigenetic Mark ◽  
Cloning Efficiency ◽  
Developmental Potential ◽  
Acetylation Status ◽  
Donor Cells ◽  
Fetal Fibroblasts

Epigenetic status of the genome of a donor nucleus is likely to be associated with the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). Prevention of epigenetic errors by manipulation of the epigenetic status of donor cells is expected to result in improvement of cloning efficiency. In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Ali/Bas) into metaphase II (MII) oocytes and analyzed the levels of histone H3K9 acetylation in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with one or two blastomeres from in vitro-fertilized or parthenogenetic embryos. Histone acetylation in donor cells and cloned embryos was detected by anti-acH3K9 antibody using Western immunoblot analysis or immunochemistry, respectively. Data were analyzed by chi-square (developmental rates) or Student-Newman-Keuls (histone acetylation) test. The levels of acetylated histone H3K9 were higher in RCCs than in RFFs (P &lt; 0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC-cloned embryos induced a higher initial pregnancy rate as compared to RFF-cloned embryos (40% vs. 20%; P &lt; 0.05). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed; a live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly (P &lt; 0.05) increased the level of histone H3K9/14 acetylation and the proportion of nuclear transfer embryos developing to blastocyst (49% vs. 33% with non-treated RFF; P &lt; 0.05). The distribution of signals for acH3K9 in either group of cloned embryos did not resemble that in in vivo-fertilized embryos, suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo-derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and can be a useful epigenetic mark to predict efficiency of SCNT rabbits. This work was supported by the Bayerische Forschungsstiftung and by Therapeutic Human Polyclonals, Inc.

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Germinal Vesicle Breakdown ◽  
Significant Difference ◽  
Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

44 IN VITRO AND IN VIVO SURVIVABILITY ON VITRIFIED EMBRYOS OBTAINED BY BOVINE SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 131
Author(s):  
K. Kaneyama ◽  
S. Kobayashi ◽  
S. Matoba ◽  
Y. Hashiyada ◽  
K. Imai ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Survival Rates ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Nuclear Transplantation ◽  
Embryo Cryopreservation

Although many studies have been conducted on somatic cell nuclear transfer, there are only a few reports on cryopreservation of reconstructed embryos after nuclear transplantation. The objective of this study was to examine in vitro or in vivo development of vitrified blastocysts obtained by nuclear transfer. Nuclear transfer was carried out according to the procedure of Goto et al. (1999 Anim. Sci. J. 70, 243–245), and conducted using abattoir-derived oocytes and cumulus cells derived by ovum pickup from Holstein and Japanese Black cows. Embryos were vitrified as described by Saito et al. (1998 Cryobiol. Cryotech. 43, 34–39). The vitrification solution (GESX solution) was based on Dulbecco's PBS containing 20% glycerol (GL), 20% ethylene glycol (EG), 0.3 M sucrose (Suc), 0.3 M xylose (Xyl), and 3% polyethylene glycol (PEG). The blastocysts were equilibrated in three steps, with 10% GL, 0.1 M Suc, 0.1 M Xyl, and 1% PEG for 5 min (1); with 10% GL, 10% EG, 0.2 M Suc, 0.2 M Xyl, and 2% PEG for 5 min (2) and GESX solution (3). After transfer to GESX, equilibrated embryos were loaded to 0.25-mL straws and plunged into liquid nitrogen for 1 min. The vitrified blastocysts were warmed in water (20°C) and diluted in 0.5 M and 0.25 M sucrose for 5 min each. Equilibration and dilution procedures were conducted at room temperature (25–26°C). After dilution, the vitrified blastocysts were cultured in TCM-199 supplemented with 20% fetal calf serum and 0.1 mM β-mercaptoethanol at 38.5°C under gas phase of 5% CO2 in air. In Experiment 1, survival rates after vitrification were compared between the nuclear transfer and the IVF blastocysts. Survival rates of vitrified nuclear transfer blastocysts (n = 60, Day 8) at 24 and 48 h were 70.0% and 56.7%, respectively, and those of vitrified IVF blastocysts (n = 41) were 82.9% and 82.9%, respectively. There were no significant differences in survival rates at 24 and 48 h between the two groups. In Experiment 2, one (VIT-single) or two (VIT-double) vitrified and one (nonVIT-single) or two (nonVIT-double) nonvitrified reconstructed blastocysts per animal were transferred into Holstein dry cows. The result of Experiment 2 is shown in Table 1. This experiment demonstrated that the vitrification method in this study can be used for cloned embryo cryopreservation but the production rate should be improved. Table 1. Comparison of survival rates of vitrified or nonvitrified cloned embryos after transfer

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