scholarly journals Effect of Cryoprotectant Composition on In Vitro Viability of In Vitro Fertilized and Cloned Bovine Embryos Following Vitrification and In-Straw Dilution

2007 ◽  
Vol 53 (4) ◽  
pp. 963-969 ◽  
Author(s):  
Masayasu TANIGUCHI ◽  
Akiko IKEDA ◽  
Eri ARIKAWA ◽  
Pimprapar WONGSRIKEAO ◽  
Budiyanto AGUNG ◽  
...  
Keyword(s):  
Bovine Embryos ◽  
Cloned Bovine

55 EFFECT OF FUSION-ACTIVATION INTERVAL AND EMBRYO AGGREGATION ON IN VITRO AND IN VIVO DEVELOPMENT OF CLONED BOVINE EMBRYOS PRODUCED BY HANDMADE CLONING

2010 ◽  
Vol 22 (1) ◽  
pp. 185
Author(s):  
R. P. C. Gerger ◽  
F. Forell ◽  
J. C. Mezzalira ◽  
F. Zago ◽  
F. K. Vieira ◽  
...  
Keyword(s):  
Somatic Cell ◽  
High Rate ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Cloned Bovine ◽  
Vitro Development ◽  
Handmade Cloning ◽  
Activation Intervals

Despite the apparent success of cloning by somatic cell nuclear transfer (SCNT), the efficiency in development to term remains low, with a high rate of losses occurring throughout pregnancy due to faulty reprogramming and conceptus abnormalities. As the ideal fusion-activation interval for optimal nuclear reprogramming after cloning is still ill-defined, the aim of this study was to determine the effect of 2 distinct fusion-activation intervals and embryo aggregation on in vitro development of cloned bovine embryos. Bovine COCs from slaughterhouse ovaries were used after IVM for the production of cloned embryos by handmade cloning, according to our established procedures (Ribeiro et al. 2009 Cloning Stem Cells, in press). Following cumulus and zona removal, oocytes were manually bisected, with hemi-cytoplasts selected by DNA staining. Two hemi-cytoplasts and an adult skin somatic cell were attached and fused with a 15V AC pre-pulse for 5 s, followed by a double 1.2 kV cm-1 DC pulse for 20 μs. Reconstructed embryos were activated in ionomycin exactly at 2 or 4 h post-fusion (2 hpf or 4 hpf), followed by an incubation in 6-DMAP for 4 h. Cloned embryos from both fusion-activation intervals were in vitro-cultured in the well of the well (WOW) system for 7 days, allocating one (1 × 100%) or two (2 × 100%) cloned embryos per WOW. Grade 1 Day-7 blastocysts were transferred to synchronous recipients. Cleavage (Day 2) and blastocyst (Day 7) rates, on a per WOW basis, and pregnancy (Days 30 and 150) rates were compared using the chi-square or the Fisher test, with results from 9 replications summarized in Table 1. Increasing the fusion-activation interval to 4 h decreased cleavage but not blastocyst rates in 1 × 100% embryos. Also, blastocyst rates were lower in 1 × 100% embryos activated 2 h post-fusion. In general, cleavage and blastocysts rates for 2 × 100% embryos (91.5 and 46.0%) were higher than for 1 × 100% embryo counterparts (74.4 and 31.3%), respectively, regardless of the activation time. In addition, blastocyst rates for 4 hpf-activated embryos (50.3%), based on cleavage, were higher than for 2 hpf-activated embryos (38.3%), irrespective of the aggregation scheme. Nonetheless, despite differences in in vitro development, pregnancy rates and conceptus development in the first half of pregnancy were similar between groups. A longer fusion-activation interval (4 hpf) or embryo aggregation (2 × 100%) increased blastocyst yield but did not improve in vivo development and pregnancy maintenance following the transfer to female recipients in cattle. Table 1.In vitro and in vivo development of cloned bovine embryos This study was supported by FAPESP and CAPES, Brazil.

scholarly journals Improved in vitro development of cloned bovine embryos using S -adenosylhomocysteine, a non-toxic epigenetic modifying reagent

2011 ◽  
Vol 78 (8) ◽  
pp. 576-584 ◽  
Author(s):  
Shahram Jafari ◽  
Morteza S. Hosseini ◽  
Mahdi Hajian ◽  
Mohsen Forouzanfar ◽  
Farnoosh Jafarpour ◽  
...  
Keyword(s):  
Bovine Embryos ◽  
In Vitro Development ◽  
Cloned Bovine ◽  
Vitro Development

scholarly journals In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence

2010 ◽  
Vol 9 (1) ◽  
pp. 295-302 ◽  
Author(s):  
R.P.C. Gerger ◽  
E.S. Ribeiro ◽  
F. Forell ◽  
L.R. Bertolini ◽  
J.L. Rodrigues ◽  
...  
Keyword(s):  
Cell Culture ◽  
Somatic Cells ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Cloned Bovine ◽  
Vitro Development ◽  
Handmade Cloning

scholarly journals Viability of cloned bovine embryos after one or two cycles of nuclear transfer and in vitro culture

1995 ◽  
Vol 44 (7) ◽  
pp. 925-933 ◽  
Author(s):  
F.J. Ectors ◽  
A. Delval ◽  
L.C. Smith ◽  
K. Touati ◽  
B. Remy ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Nuclear Transfer ◽  
Bovine Embryos ◽  
Cloned Bovine

38 EFFECTS OF TRICHOSTATIN A ON DNA METHYLATION IN CLONED BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 99 ◽  
Author(s):  
D. Iwamoto ◽  
S. Kishigami ◽  
S. Taniguchi ◽  
Y. Abe ◽  
T. Matsui ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Trichostatin A ◽  
Calcium Ionophore ◽  
Serum Starvation ◽  
Image Analyzer ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Cloned Bovine

Recently, the efficiency of full-term development of somatic cloned mouse embryos was significantly increased by treatment with trichostatin A (TSA), an inhibitor of histone deacetylase (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). We have shown that TSA treatment improved the rate of development of the cloned bovine embryos to the blastocyst stage (Iwamoto et al. 2007 Reprod. Fertil. Dev. 19, 142 abst). Higher levels of DNA methylation have been shown in early cloned bovine embryos than in in vitro-fertilized (IVF) embryos (Dean et al. 2001 Proc. Nat. Acad. Sci. USA 98, 13734–13738; Santos et al. Curr. Biol. 13, 1116–1121). In this study, we examined the effects of TSA on DNA methylation levels in cloned bovine embryos by immunostaining with an antibody to 5-methyl cytosine (5-MeC). Bovine fibroblasts were cultured under serum starvation (0.4% FCS) for 7 days before they were used as donor cells. The cells were electrofused with bovine enucleated matured oocytes, and activated with a calcium ionophore and cycloheximide. Atotal of 131 cloned embryos were produced. The NT embryos were exposed to 0 (control) and 50 nmTSA from the start of activation to 48 h post-activation (hpa). They were then cultured in an mSOF medium. At 60 hpa, only embryos developed to the 8-cell stage were used for assessment of DNA methylation levels. Sixteen TSA-treated, 22 non-treated, and 19 IVF embryos were immunostained with 5-MeC antibody. For quantitative analysis of the DNA methylation levels, 5-MeC signals in the fluorescent images were determined using an image analyzer system (Aqua Cosmos; Hamamatsu Photonics, Shizuoka, Japan). The data were analyzed with Tukey-Kramer post hoc test for multiple comparisons following ANOVA. Relative levels of DNA methylation of TSA-treated cloned and IVF embryos did not differ (P > 0.05), but were lower than those of non-treated cloned embryos (P < 0.05). The results indicate that TSA treatment of cloned bovine embryos leads to a reduction of DNA methylation levels of their genome. The data suggest that the TSA treatment decreased the DNA methylation levels of cloned bovine embryos to the levels of IVF embryos, resulting in improved blastocyst development of the cloned embryos.

Improving in vitro development of cloned bovine embryos with hybrid (Holstein–Chinese Yellow) recipient oocytes recovered by ovum pick up

2005 ◽  
Vol 64 (6) ◽  
pp. 1263-1272 ◽  
Author(s):  
Xiao-Yu Yang ◽  
Jian-Guo Zhao ◽  
Hua-Wei Li ◽  
Hua Li ◽  
Hai-Feng Liu ◽  
...  
Keyword(s):  
Bovine Embryos ◽  
In Vitro Development ◽  
Cloned Bovine ◽  
Vitro Development

Effects of donor cells on in vitro development of cloned bovine embryos

2008 ◽  
Vol 35 (5) ◽  
pp. 273-278 ◽  
Author(s):  
Jing Fu ◽  
Pengfei Guan ◽  
Leiwen Zhao ◽  
Hua Li ◽  
Shuzhen Huang ◽  
...  
Keyword(s):  
Bovine Embryos ◽  
In Vitro Development ◽  
Donor Cells ◽  
Cloned Bovine ◽  
Vitro Development

Post-thaw in vitro survival of vitrified cloned bovine embryos

1997 ◽  
Vol 140 (15) ◽  
pp. 404-404 ◽  
Author(s):  
P. J. Booth ◽  
G. Vajta ◽  
P. Holm ◽  
T. Greve ◽  
H. Callesen
Keyword(s):  
Bovine Embryos ◽  
Cloned Bovine ◽  
In Vitro Survival

23 EFFECT OF HISTONE ACETYLATION LEVELS ON THE IN VITRO DEVELOPMENT OF CLONED BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 111
Author(s):  
L. Chacón ◽  
J. A. Jenkins ◽  
S. P. Leibo ◽  
G. Wirtu ◽  
B. L. Dresser ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Cultured Cells ◽  
Pearson Correlation ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Donor Cells ◽  
Cloned Bovine ◽  
Vitro Development

The epigenetic status of donor cells is an important factor for their successful reprogramming during somatic cell nuclear transfer (SCNT). Environmental factors partly influence DNA methylation and histone modifications (Fraga et al. 2005 PNAS USA 102, 10 604–10 609; Ke et al. 2006 Carcinogenesis 27, 1481–1488; Rodenhiser and Mann 2006 CMAJ 174, 341–348); low temperatures have altered epigenetic events in plants (Amasino 2004 Plant Cell; Hao et al. 2002 Cryo Letters 23, 37–46). Because cryopreservation alters histone acetylation levels in donor cells and subsequent viability of cloned embryos (Gómez et al. 2008 Cloning Stem Cells, in press), similar alterations may occur in bovine cloned embryos reconstructed with donor cells thawed immediately before SCNT. The objectives of the present study were (1) to measure the relative levels of nuclear histone acetylation in bovine fibroblasts immediately after thawing (frozen/thawed) or following a period of culturing (cultured) and (2) to determine the influence of the epigenetic status of donor cells on the in vitro development of reconstructed, cloned bovine embryos by gauging blastocyst development. Cell cultures lines were derived from the skin of 3 adult cows and analyzed at passage 1 (P1), 2 (P2), and 10 (P10). For each of 3 passages, cells were cultured until reaching 100% confluence, followed by an additional 3 days of culture during which time acetylation levels were measured in cultured and frozen/thawed cells. For cryopreservation, cells at P1, P2, and P10 were disaggregated and resuspended in CryoStor™ (CS10; BioLife Solutions, Bothell, WA, USA) and cooled at 1.0°C min–1 to –80°C prior to storage in liquid nitrogen. Cells were fixed with ethanol for 12 h and incubated for 30 min with antibody directed against acetylated lysine 9 on histone 3 (H3K9). The cells were then incubated with a fluorescein isothiocyanate conjugated secondary antibody and DNA stain and evaluated by flow cytometry. Cloned embryos were reconstructed with cultured or frozen/thawed cells at P1, P2, and P10 as described by Vajta et al. 2005 (Reprod. Fertil. Dev. 17, 791–797). Derived embryos were cultured until Day 8, and cleavage and development to the blastocyst stage were evaluated. Histone acetylation levels for all 3 cell lines, either fresh or frozen/thawed, were significantly higher at P1 than at P2 and P10 (Table 1), and cryopreservation reduced histone acetylation levels only in cell culture line 2 at P1. Higher development to the blastocyst stage (25%) was observed when embryos were reconstructed with cultured cells at P2 and with cells that had lower histone acetylation levels (Pearson correlation, r = –0.55; P = 0.01) Table 1.Relative levels of histone acetylation in bovine fibroblast culture and percentages of development to blastocyst stage after cloning

Sign in / Sign up

Export Citation Format

Share Document