Effect of Cytoplasmic Volume on Developmental Competence of Buffalo (Bubalus bubalis) Embryos Produced Through Hand-Made Cloning

2011 ◽  
Vol 13 (3) ◽  
pp. 257-262 ◽  
Author(s):  
Sudeepta K. Panda ◽  
Aman George ◽  
Ambika P. Saha ◽  
Ruchi Sharma ◽  
Radhey S. Manik ◽  
...  
Keyword(s):  
Bubalus Bubalis ◽  
Developmental Competence

65 EFFECT OF TRICHOSTATIN A AND 5-AZA-2'-DEOXYCYTIDINE TREATMENT OF DONOR CELLS, FUSED EMBRYOS, OR BOTH ON THE DEVELOPMENTAL COMPETENCE, QUALITY, AND EPIGENETIC STATUS OF CLONED BUFFALO (BUBALUS BUBALIS) EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 162
Author(s):  
M. Saini ◽  
N. L. Selokar ◽  
H. Agrawal ◽  
S. K. Singla ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Species Conservation ◽  
Bubalus Bubalis ◽  
Cell Number ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Epigenetic Modifiers ◽  
Donor Cells ◽  
Significant Difference ◽  
Transgenic Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology in buffalo for multiplication of elite animals, species conservation, and production of transgenic embryos for therapeutic applications. However, the cloning efficiency obtained in this species is very low, which might be due to improper reprogramming of donor cells after SCNT. Treatment of donor cells or fused embryos or both with epigenetic modifiers might be a suitable approach to improve the ability of donor cells to be reprogrammed. The present study was aimed at examining the effects of treatment of donor cells (24 h before SCNT) or fused embryos (10 h post-electrofusion) or both with 50 nM TSA + 7.5 nM 5-aza-dC on the developmental competence, quality, and epigenetic status of buffalo embryos produced by hand-made cloning (HMC) as described earlier (Saini et al. 2014 Reprod. Fertil. Dev. doi: 10.1071/RD14176). The percentage data were analysed using SYSTAT 12.0 (SPSS Inc., Chicago, IL, USA) after arcsine transformation. Differences between means were analysed by one-way ANOVA followed by Fisher’s least significant difference test. The blastocyst rate was significantly higher (P < 0.05) and the apoptotic index was significantly lower (P < 0.05) in embryos produced from donor cells or fused embryos or both treated with TSA + 5-aza-dC than that of controls (Table 1). However, the cleavage rate and the total cell number were not significantly different among all the groups. The global level of H3K18ac, examined by immunofluorescence staining, was higher (P < 0.05) and that of H3K27me3 was lower (P < 0.01) in blastocysts produced from donor cells or fused embryos or both treated with TSA + 5-aza-dC than that of controls. These results show that treatment of donor cells, fused embryos, or both with TSA + 5-aza-dC improves the developmental competence and quality, and alters the epigenetic status of buffalo embryos produced by HMC. However, the effects of treatment with these epigenetic modifiers on the pregnancy rate require further studies. Table 1.Effect of treatment of donor cells, fused embryos, or both with 50 nM TSA + 7.5 nM 5-aza-dC on the developmental competence and level of apoptosis in cloned embryos

Effect of DNA Methylation Inhibitors on the Developmental Competence of Cloned Buffalo (Bubalus bubalis) Embryos.

2011 ◽  
Vol 85 (Suppl_1) ◽  
pp. 781-781 ◽  
Author(s):  
Ambikaprasanna Saha ◽  
Naresh Lalaji Selokar ◽  
Sudeepta Kumar Panda ◽  
Aman George ◽  
Musharifa Muzaffar ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bubalus Bubalis ◽  
Developmental Competence ◽  
Dna Methylation Inhibitors

Early Cleavage of Handmade Cloned Buffalo (Bubalus bubalis ) Embryos is an Indicator of Their Developmental Competence and Quality

2015 ◽  
Vol 50 (2) ◽  
pp. 214-220 ◽  
Author(s):  
S Kaith ◽  
M Saini ◽  
AK Raja ◽  
AA Sahare ◽  
B Jyotsana ◽  
...  
Keyword(s):  
Bubalus Bubalis ◽  
Developmental Competence ◽  
Early Cleavage

134 EFFECTS OF VITAMIN E AND VITAMIN C ON THE DEVELOPMENTAL COMPETENCE OF BUFFALO (BUBALUS BUBALIS) EMBRYOS DERIVED FROM PARTHENOGENETIC ACTIVATION, IN VITRO FERTILIZATION, AND NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 171
Author(s):  
F. Lu ◽  
Z. Zhang ◽  
S. Zhang ◽  
N. Li ◽  
J. Jiang ◽  
...  
Keyword(s):  
Vitamin E ◽  
Nuclear Transfer ◽  
Bubalus Bubalis ◽  
Development Rate ◽  
The Other ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Parthenogenetic Activation

The purpose of this study was to explore the effects of vitamin E (VE) and vitamin C (VC) on the in vitro development of embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF), and somatic cell nuclear transfer (NT) in buffalo (Bubalus bubalis). Buffalo oocytes obtained from ovaries at slaughter were matured in vitro for 22 to 24 h. After maturation, oocytes were separated to 3 groups: one group of oocytes was fertilized in vitro with buffalo sperm; one group of oocytes was parthenogenetically activated by exposing them to 5 μM ionomycin for 5 min and then cultured in 2 mM 6-DMAP for 3 h; the other group of oocytes was enucleated, and fibroblasts in DMEM + 10% FBS for 4 to 5 days were transferred into enucleated oocytes by electronic fusion (100 v mm–1, 15 μs, and 3 pulses). After fusion, the activation of reconstructed embryos was induced by exposure to 5 μM ionomycin for 5 min and then cultured in 2 mM 6-DMAP for 3 h. The embryos of PA, IVF, and NT were respectively cultured in the culture medium (CM) containing different concentrations of VE, VC, or VE + VC for 7 to 9 days to evaluate embryonic development. As a result, when the embryos were cultured in the CM with different concentrations of VE (0, 50, 100, 150, and 200 μM), the blastocyst development rate of the embryos derived from PA, IVF, and NT gradually rose with increasing concentrations of VE and reached the highest amount [PA: 32.9% (81/246); IVF: 21.4% (45/210); and NT: 21.1% (47/223)] in the group containing 150 μM of VE; it was significantly higher than that of other groups (P < 0.05). When the different concentrations of VC (0, 50, 100, 150, and 200 μM) were added to the CM, the blastocyst development rate of the embryos derived from PA, IVF, and NT also enhanced according to the increasing concentration of VC, and more embryos developed to blastocysts in the group containing 150 μM of VC [PA: 31.2% (72/231); IVF: 20.2% (43/213); NT: 19.8% (48/243)] than in the other groups (P < 0.05). Compared with the control group (0 μM), the blastocyst rate of PA and IVF, as well as NT embryos, cultured in the CM with 150 μM VE + 150 μM VC groups was significantly higher (P < 0.05), but there were no significant differences in the percentage of blastocysts among groups of the 150 μM VE, 150 μM VC, and 150 μM VE + 150 μM VC (P > 0.05). These results indicated that adding VE (150 μM), VC (150 μM), or VE (150 μM) + VC (150 μM) in the CM could efficiently enhance the developmental competence of buffalo embryos during in vitro culture. This work was funded by China High Technology Development Program (2007AA100505), Guangxi Science Foundation (0718005-3A), Fok Ying Tung Education Foundation (111034).

60 EFFECT OF CYTOPLASMIC VOLUME ON DEVELOPMENTAL COMPETENCE OF HAND-GUIDED CLONED BUFFALO (BUBALUS BUBALIS) EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 135
Author(s):  
S. K. Panda ◽  
A. George ◽  
A. P. Saha ◽  
R. Sharma ◽  
N. M. Kamble ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryonic Stem ◽  
Cell Types ◽  
Bubalus Bubalis ◽  
Developmental Competence ◽  
Cloning Efficiency ◽  
Cleavage Rate ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate

Despite recent successes in the birth of buffalo calves cloned through SCNT or hand-guided cloning (HGC), the cloning efficiency is very low in this species because of lack of information on factors that influence it. The goal of this study was to examine the effects of cytoplasmic volume on the developmental competence of cloned buffalo embryos produced by HGC. In vitro matured oocytes were stripped of their cumulus investment and zona pellucida using hyaluronidase and pronase, respectively. Protrusion cone-guided bisection of zona-free oocytes was performed to remove the nucleus. For reconstructing control HGC embryos, 2 enucleated oocytes (demi-cytoplasts) were fused with a single somatic cell. For reconstruction of embryos with lower or higher cytoplasmic volume, 1 or 3 demi-cytoplasts were fused, respectively, with the donor somatic cell. 2 different cell types, i.e. buffalo fetal fibroblasts (BFF) between passage 10 and 15 and buffalo embryonic stem cell (ESC)-like cells between passage 22 and 25 were used as nuclear donors in 2 different experiments. Data were analysed by 1-way ANOVA after arcsine transformation of percentage values. For BFF, the blastocyst rate for doublet and triplet embryos were significantly higher (P ≤ 0.01) than that for singlet embryos despite the cleavage rate for the 3 groups being similar. For the ESC-like cells, the cleavage and the blastocyst rate were significantly lower (P ≤ 0.01) for the singlet than that for the doublet embryos. The pregnancies were established only in doublet and triplet embryo groups using BFF cells and in the doublet embryo group using ESC-like cells. These results indicate that increasing the cytoplasmic volume could be helpful in improving cloning efficiency in terms of blastocyst production rate in buffaloes. Table 1.Effect of cytoplasmic volume on the developmental competence of cloned buffalo embryos This work was funded by NAIP grant C 2-1-(5)/2007 to SKS.

Low oxygen tension improves developmental competence and reduces apoptosis in hand-made cloned buffalo (Bubalus bubalis) embryos

2015 ◽  
Vol 172 ◽  
pp. 106-109 ◽  
Author(s):  
M. Saini ◽  
N.L. Selokar ◽  
H. Agrawal ◽  
S.K. Singla ◽  
M.S. Chauhan ◽  
...  
Keyword(s):  
Oxygen Tension ◽  
Bubalus Bubalis ◽  
Developmental Competence ◽  
Low Oxygen ◽  
Low Oxygen Tension
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