76 PRODUCTION OF CLONED BOVINE TRANSGENIC EMBRYOS WITH VARIOUS TYPES OF MONO-COLONY CELLS AND OVUM PICKUP

2005 ◽  
Vol 17 (2) ◽  
pp. 188
Author(s):  
J.G. Zhao ◽  
X.Y. Yang ◽  
Y. Huang ◽  
H.F. Liu ◽  
H. Li ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Nuclear Transfer ◽  
Genetic Manipulation ◽  
Blastocyst Stage ◽  
High Tech ◽  
Cleavage Rate ◽  
Exogenous Dna ◽  
Developmental Potential ◽  
Reconstructed Embryos

The objective of this study was to determine the effects of genetic manipulation, cell type, and culture conditions on developmental potential of bovine nuclear transfer (NT) embryos. Ovum pickup (OPU) technology was developed to obtain the oocytes for NT. A total 4044 cumulus-oocyte complexes (COCs) were obtained during 492 OPU sessions, with an average of 8.2 COCs recovered each session. Cultured granulosa cells (CGC), bovine fetal (150 days) oviduct epidermic cells (FOEC), and adult ear skin fibroblasts (ASFC) were used as donor cells for NT and were transfected with the expression vector including human FIX coding sequence directed by goat β-casein promoter and neomycin gene. The cells were screened under 800 μg mL−1 G418 for 10–14 days until the apperance of a “mono-colony” of cells which were then picked. Each cell population was expanded by consecutive passage culture under 300 μg mL−1 G418 until used for NT, ensuring that the majority of cells were transgenic. Oocytes were enucleated at 20 h post-maturation and a single donor cell was transferred into the perivitelline space of a recipient oocyte. After fusion and activation, the reconstructed embryos were co-cultured with vero cells in B2 medium for 7 days. NT efficiency between primary granulosa cells (PGC) without in vitro culture and CGC, as well as among CGC, FOEC and ASFC that were transfected with exogenous DNA (named TCGC, TFOEC, TASFC, respectively), were compared (Table 1). Differences between groups were verified by chi-square test using SAS 6.12 (SAS Institute, Inc., Cary, NC, USA) program. CGCs presented a higher fusion rate (P < 0.01) for reconstructed embryos and higher development to the blastocyst stage for NT embryos than did PGC (67% vs. 54% and 41% vs. 21%, respectively). There were no significant differences (P > 0.05) in cleavage rate (65%, 71%, and 69%, respectively) and development to the blastocyst stage for NT embryos (36%, 30% and 40%, respectively) for TCGC, TFOEC, and TASFC. A total of 86 blastocysts were selected for transfer into uteri of 86 cows, resulting in 26 pregnancies (30%) at 60 days by ultrasound scanning. Among these, 12 cows remain pregnant and 14 have aborted. The results indicated that oocytes recovered from OPU can be successfully used for NT with development to the blasocyst stage. PGC, CGC, FOEC, and ASFC can all be used for generating transgenic cattle by NT, although this needs to be verified by the birth of live calves. Table 1. Nuclear transfer efficiency with various cell types This work was supported by the Chinese “863” High-Tech Plan Program (Grant No. 2002AA206201).

58 HISTONE DEACETYLASES INHIBITOR, SCRIPTAID, IS BENEFICIAL TO THE REPROGRAMMING OF SOMATIC NUCLEI FOLLOWING NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 129
Author(s):  
J. G. Zhao ◽  
J. W. Ross ◽  
Y. H. Hao ◽  
D. M. Wax ◽  
L. D. Spate ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylases ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Untreated Group ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology with potential applications in both agriculture and regenerative medicine. The reprogramming of differentiated somatic nuclei into totipotent embryonic state following NT is not efficient and the mechanism is currently unknown. However, accumulating evidence suggests that faulty epigenetic reprogramming is likely to be the major cause of low success rates observed in all mammals produced through SCNT. It has been demonstrated that increased histone acetylation in reconstructed embryos by applying histone deacetylases inhibitor (HDACi) such as trychostatin A (TSA) significantly enhanced the developmental competence in several species in vitro and in vivo. However TSA has been known to be teratogenic. Compared with TSA, Scriptaid is a low toxic but more efficient HDACi (Su GH et al. 2000 Cancer Res. 60, 3137–3142). The objectives of this study were: 1) to investigate and optimize the application Scriptaid to the NT using Landrace fetal fibroblast cells (FFCs) as donor; 2) investigate the effect of increased histone acetylation on the developmental competence of reconstructed embryos from NIH mini inbred FFCs in vitro and in vivo. The reconstructed embryos were treated with Scriptaid at different concentrations (0 nm, 250 nm, 500 nm and 1000 nm) after activation for 14 to 16 h. IVF embryos without treatment were produced as an additional control. Developmental rates to the 2-cell and blastocyst stage were determined. Developmental potential was determined by transferring Day 1 NT zygotes to the oviducts of surrogates on the day of, or one day after, the onset of estrus. Experiments were repeated at least 3 times and data were analyzed with chi-square tests using SAS 6.12 program (SAS institute, Inc., Cary, NC, USA). The percentage blastocyst of cloned embryos using Landrace FFCs as donors treated with 500 nm Scriptaid was the highest and was significantly higher than untreated group (25% v. 11%, P < 0.05). Percent cleaved was not different among four treatment groups. We used 500 nm Scriptaid for 14 to 16 h after activation for all subsequent experiments. Developmental rate to the blastocyst stage was significantly increased in cloned embryos derived from NIH mini inbred FFCs after treating with Scriptaid (21% v. 9%, P < 0.05), while the blastocyst rate in IVF group was 30%. Embryo transfer (ET) results showed that 5/6 (Transferred embryos No. were 190, 109, 154, 174, 152, and 190, respectively) surrogates (83%) became pregnant resulting in 2 healthy piglets from 2 litters (recipients received 190 and 154 embryos, respectively) in the Scriptaid treatment group, while no pregnancies were obtained in the untreated group from 5 ET (Embryos transferred No. are 140, 163, 161, 151 and 151, respectively). These results suggest that 500 nm Scriptaid treatment following activation increase both the in vitro and in vivo development of porcine SCNT embryos from NIH mini inbred FFCs and the hyperacetylation might actually improve reprogramming of the somatic nuclei after NT. Funding from the National Institutes of Health National Center for Research Resources RR018877.

55 PRELIMINARY STUDIES ON THE DEVELOPMENT OF RECONSTRUCTED EMBRYOS AFTER NUCLEAR TRANSFER IN DROMEDARY CAMEL (CAMELUS DROMEDARIUS)

2009 ◽  
Vol 21 (1) ◽  
pp. 128 ◽  
Author(s):  
N. A. Wani ◽  
J. A. Skidmore ◽  
U. Wernery
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Uv Light ◽  
Light Exposure ◽  
Blastocyst Stage ◽  
Dromedary Camel ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Reconstructed Embryos

Experiments were conducted to study the in vitro development of reconstructed dromedary camel embryos after nuclear transfer by a modified zona-free method. Cumulus oocyte complexes, collected from slaughterhouse ovaries were cultured in TCM199 at 38.5°C in an atmosphere of 5% CO2 in air for 32 to 36 h. Matured oocytes were denuded of cumulus cells by repeated pipetting and the zona pellucida was removed by brief incubation in 5 mg mL–1 pronase dissolved in Ca- and Mg-free PBS. Zona-free oocytes were stained with 5 mg mL–1 Hoechst 33342 in H199 supplemented with 7.5 μg mL–1 cytochalasin B and 10% FCS. They were enucleated under constant UV-light exposure in H199 supplemented with cytochalasin B and 10% FCS. The granulosa cells at passage numbers 4 to 15 were used as nuclear donors. The zona-free cytoplasts were individually washed for a few seconds in 300 μg mL–1 of Phytohemagglutinin in H199, then quickly dropped on a single donor cell settled to the bottom of a drop of H199 with 0.5% FCS and pushed together with the mouth pipette. Couplets were electrically fused, at room temperature, with two DC pulses of 100 V cm–1 for 15 μs. Reconstructs were activated 2 h post-fusion, with 5 μm ionomycin for 3 min followed by culture in 6-diethylaminopurine for 4 h. The reconstructs were then cultured individually in either 5 μL drops under oil, in agar wells or in wells of wells (WOW) in a well of 4-well culture plate. Embryo culture medium consisted of TCM-199 supplemented with 0.15 mg mL–1 L-glutamine, 2.1 mg mL–1 sodium bicarbonate, 0.22 mg mL–1 pyruvate, 50 μg mL–1 gentamycine, 1% insulin-transferrin-selenium (ITS), and 15% estrous dromedary serum. The number of oocytes that had cleaved was recorded on day 2, whilst those developing to morulae and blastocysts were recorded on day 7 of culture. For cell count, the blastocysts were stained with Hoechst and cells counted under a fluorescent microscope at ×400. Data obtained was analysed by chi-square test. About 92% (349/380) of the oocytes were successfully enucleated and 76% (259/340) fused with the attached cells. The cleavage rate was significantly lower (P < 0.05) in reconstructed embryos cultured in droplets (10/72, 14%) as compared with those cultured in agar wells (37/87, 42%) or WOW system (42/96, 44%). The proportions of cleaved embryos reaching morula stage were 0, 83, and 89% in droplets, agar wells, and WOW, respectively. However, only 8% and 5% of the cleaved embryos developed to the blastocyst stage in the agar well and WOW culture systems, respectively. No difference was observed in the cell number of blastocysts produced in agar wells (77.3 ± 8.02) or WOW (78.0 ± 4.2) culture system. To the best of our knowledge, this is the first report of embryo production up to the blastocyst stage after NT in camelids and it shows that NT can be successfully applied for embryo production in camelids. Further studies are needed to optimize the parameters and to improve the efficiency for production of transferable blastocysts in this species. This study was kindly sponsored by H.H. General Sheikh Mohammed bin Rashid Al Maktoum, Ruler of Dubai.

45 IN VITRO DEVELOPMENT OF BOVINE TRANSGENIC NUCLEAR TRANSFER EMBRYOS IN SERUM-FREE AND SERUM-SUPPLEMENTED MEDIA

2011 ◽  
Vol 23 (1) ◽  
pp. 128
Author(s):  
J. Y. Lee ◽  
S. G. Lee ◽  
E. J. Jung ◽  
S. H. Jeong ◽  
C. J. Yang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cmv Promoter ◽  
Developmental Potential ◽  
Serum Free ◽  
Reconstructed Embryos ◽  
Free Media

Novel serum-free media (IVD101) has been shown to be effective for the production of in vitro-produced embryos for subsequent implantation into cows (Hoshi 2003 Theriogenology 59, 675–685). The objective of the present study was to determine whether serum-free embryo cultivation during preimplantation stage could be used for the production of bovine transgenic nuclear transfer embryos. Somatic cell nuclear transfer (SCNT) embryos were produced by using donor cells containing a vector to induce the production of human erythropoietin in cow's milk. αS1-casein was selected as the promoter to be used in this study through the specific promoter activity test, and enhanced green fluorescent protein(EGFP) gene was attached to the CMV promoter to allow observation of the donor cell during the experiment. Adult fibroblast cells were transfected with lipofectamine. After G418 selection, the transfected cells were injected to the enucleated oocytes, and injected embryos were accomplished by cell-to-cell fusion. These embryos were then activated with calcium ionomycin and 6-dimethylaminopurine. The reconstructed embryos were cultured in IVD101 and mSOF media at 38.5°C, in a 5% CO2, 5% O2, and 90% N2 atmosphere. Embryos were cultured for 4 days, followed by addition of FBS in case of mSOF media. On day-7, the developmental ability and the number of cells in the reconstructed embryos were determined. Statistical analysis of embryo development data was carried out using unpaired t-test, or ANOVA. There were no significant differences in the cleavage rate (69.6 ± 3.2% v. 64.5 ± 5.0%), blastocyst rate (18.7 ± 1.3% v. 22.0 ± 1.6%), and cell number (113.9 ± 7.5 v. 103.6 ± 7.9) between IVD101 and mSOF+FBS cultured embryos. These results indicated that serum-free media did not reduce the developmental competence of SCNT embryos compared with serum-supplemented media. Further studies are required to investigate whether this serum-free transgenic embryo cultivation could be used for developmental potential in terms of full-term development after embryo transfer.

scholarly journals 324SPHINGOSINE-1-PHOSPHATE PROTECTS CULTURED BOVINE OOCYTES FROM PHYSIOLOGICALLY RELEVANT THERMAL STRESS

2004 ◽  
Vol 16 (2) ◽  
pp. 282 ◽  
Author(s):  
Z. Roth ◽  
P.J. Hansen
Keyword(s):  
Thermal Stress ◽  
Heat Shock ◽  
Blastocyst Stage ◽  
Caspase Activity ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
Oocyte Competence ◽  
Cell Death Pathway

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that can block the sphingomyelin cell-death pathway by suppressing ceramide-induced apoptosis. The present study was performed to test whether S1P protects oocytes from heat shock during in vitro maturation. Cumulus-oocyte complexes obtained by slicing follicles were placed in maturation medium with or without 50nM S1P and cultured at 38.5°C (CON) or 41°C (41C) for the first 12h of maturation. Incubation during the last 10h of maturation (22-h total maturation time), fertilization, and embryonic development were performed at 38.5°C and 5% (v/v) CO2. Blastocyst development was recorded at 8 days post-insemination (dpi) and activity of group II caspases in 8-day blastocysts was determined using a fluoroprobe, PhiPhiLux-G1D2 (OncoImmunin, Gaithersburg, MD, USA). Data were analysed by least-squares ANOVA with the GLM procedure of SAS. Percentage data were subjected to arcsin transformation before analysis. Exposure of oocytes to thermal stress during the first 12h of maturation reduced cleavage rate (P&lt;0.01) and the number of oocytes developing to the blastocyst stage (P&lt;0.04). There was a temperature x S1P interaction for cleavage rate (P&lt;0.03) because S1P blocked effects of thermal stress on cleavage rate. Without S1P, the percentage of oocytes that cleaved by 3 dpi were 83.6±2.7% and 65.8±2.7% for CON and 41C, respectively. In the presence of S1P, percent cleavage was 86.7±2.7% and 83.9±2.7% for CON and 41C, respectively. There was a trend (P=0.06) for a temperature x S1P interaction for percent oocytes developing to blastocyst stage because S1P blocked effects of heat shock on development. Without S1P, the percentages of oocytes that developed to the blastocyst stage were 28.7±3.0% and 15.2±3.0% for CON and 41C, respectively. In the presence of S1P, percent blastocysts were 24.3±3.4% and 23.9±3.0% for CON and 41C, respectively. When development was expressed as percentage of cleaved embryos, however, there were no effects of temperature, S1P, or temperature x S1P on percent development to the blastocyst stage. Blastocyst caspase activity was not affected by temperature or S1P. In summary, exposure to physiologically relevant thermal stress during the first 12h of maturation has a deleterious effect on oocyte competence and this effect can be reduced by S1P. The fact that heat shock reduced the percentage of oocytes but not the percentage of cleaved embryos that became blastocysts suggests that oocytes that survive effects of heat shock and cleave have normal potential to develop to the blastocyst stage. Moreover, since heat shock did not affect caspase activity, it is likely that blastocysts from heat-shocked oocytes have normal developmental potential, at least as determined by caspase activity. Support: BARD FI-330-2002 and USDA Grants 2002-35203-12664 and 2001-52101-11318.

scholarly journals 64COMPARISON OF DEVELOPMENTAL POTENTIAL OF IN VIVO AND IN VITRO RECIPIENT OOCYTES AFTER NUCLEAR TRANSFER IN GOAT

2004 ◽  
Vol 16 (2) ◽  
pp. 154
Author(s):  
H.S. Park ◽  
M.Y. Lee ◽  
S.P. Hong ◽  
J.I. Jin ◽  
J.K. Park ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Serum Starvation ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

Recent techniques in somatic cell nuclear transfer (SCNT) have been widely used for animal research. In addition, SCNT techniques may allow for the rescue of endangered species. Despite efforts for wildlife preservation, however, some threatened or endangered wild animal species will likely become extinct. As a preliminary experiment of a series in wildlife research, we tried to identify an improved method for the production of more transferable NT embryos in goats. Mature donor animals of Korean native goats (20–25kg) were synchronized with a CIDR (type G; InterAg, New Zealand) vaginal implant for 10 days followed by a total of 8 twice daily injections of 70mg of FSH (Folltropine, London, Ontario, Canada) and 400IU of hCG (Chorulon, Intervet, Moxmeer, The Netherlands). Oocytes were then collected surgically by retograde oviduct flush or direct aspiration from ovarian follicles in vivo at 29–34h after hCG. Oocytes collected from follicles were matured in TCM-199 containing 10% FBS and hormones. Prepared ear skin cells from the goat were cultured in TCM-199 containing 10% FBS at 39°C, 5% CO2 in air, and confluent monolayers were obtained. Oocytes were enucleated and donor cells from serum starvation (0.5%) culture were fused through a single electric pulse (DC 2.36kvcm−1, 17μs), and then activated by a single electric pulse (AC 5vmm−1, 5s+DC 1.56kvcm−1, 30μs) or chemical treatment (5μgmL−1 ionomycin 5min−1, 1.9mM 6-DMAP/4h). Reconstructed oocytes were cultured in M16 medium with 10% goat serum (GS) for 6–7 days. Data were analyzed by chi-square test. In in vitro development, significantly (P&lt;0.05) more oocytes were cleaved (24/30, 80.0%) and developed (7/24, 29.2%) to morula or blastocyst stage, respectively, in NT oocytes activated by Iono + DMAP compared to electric stimulated oocytes (2/21, 40.0%; 0/2, 0%). There was a significant difference in in vitro development of NT embryos by the method of oocyte collection. Cleavage rate was higher (P&lt;0.05) in NT embryos from in vivo oocytes (23/28, 82.1%) than in in vitro matured oocytes (19/35, 54.3%), and further development to morula or blastocyst was also significantly (P&lt;0.05%) higher in NT embryos from in vivo oocytes (7/23, 30.4%) than in NT embryos from in vitro matured oocytes (0/19, 0%). When we compared NT embryos to parthenotes, developmental rate was not significantly different between NT embryos and parthenotes. These results strongly suggest that the in vivo oocytes will have superior developmental potential to oocytes matured in vitro. Table 1 Effect of different oocyte source on in vitro development following caprine SCNT

29 EVALUATION OF DIFFERENT FUSION PARAMETERS IN THE RECONSTRUCTION OF SWINE HANDMADE CLONING EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 95
Author(s):  
C. Feltrin ◽  
A. S. Lima ◽  
M. Monaco ◽  
S. M. Wilson ◽  
D. Kim ◽  
...  
Keyword(s):  
Uv Light ◽  
Polar Body ◽  
Donor Cell ◽  
Fusion Rate ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Reconstructed Embryos ◽  
Cloning Technique ◽  
Handmade Cloning

The goal of this experiment was to compare different fusion parameters in the handmade cloning technique to produce cloned swine embryos. After in vitro maturation of 618 oocytes, 431 (69.8%) presented a visible polar body and were used in the experiment. The next step was the removal of the cumulus oophorus cells and the digestion of the zona pellucida using pronase (5 mg mL–1) in HEPES TCM199. Oocytes were then exposed to a medium containing cytochalasin B (5 µg mL–1) for 15 min before being bisected with a hand-held blade. The bisected oocytes (cytoplasts) were then placed in medium supplemented with Hoechst 33342 and exposed to UV light to select cytoplasts without metaphase II plates. Next, two cytoplasts and a mesenchymal stem cell (nucleus donor) were pushed together in a phytohemagglutinin (550 µg mL–1) solution. Once adhered, these structures were divided into 3 groups (G) to be fused using different parameters: (G1) 2 pulses (DC) of 0.6 kV cm–1 for 30 µs, (G2) 2 pulses (DC) of 0.9 kV cm–1 for 30 µs, and (G3) 2 pulses (DC) of 1.2 kV cm–1 for 30 µs. For all three groups, 0.3 m of mannitol solution (without calcium) was used in the fusion chamber, and an initial pre-pulse (AC) of 10V for 15 s was performed to permit the alignment of 100% of the cytoplast-donor cell structures. After fusion, reconstructed embryos were activated in 0.3 m mannitol and 0.1 mm calcium in the fusion chamber using 2 pulses of 0.9 kV cm–1 for 30 µs followed by incubation in 10 µg mL–1 of cycloheximide solution for 4 h. Afterwards, the reconstructed embryos were transferred to NCSU23 medium supplemented with amino acids (nonessential and essential) and 0.4% bovine serum albumin. The embryos were cultured at 39�C in a 100% humidified atmosphere containing 5% CO2, 5% O2, and 90% N2. Cleavage rates were evaluated after 48 h of culture. For G1, the fusion rate was 43% (25/58) with 72% cleavage (18/25), the G2 fusion rate was 87% (56/64) with 80% cleavage (45/56), and the G3 fusion rate was 79% (53/67) with 69% cleavage (37/53). Statistical analysis was performed using the chi-square test. There were no significant differences in fusion rates between groups G2 and G3, but the fusion rate of these groups was significantly different from that of G1 (P < 0.05). No significant differences in cleavage rate were observed among the three groups. In conclusion, fusion using 2 pulses at either 0.9 or 1.2 kV cm–1 for 30 µs was more efficient for embryo reconstruction in the handmade cloning technique compared to that using 2 pulses at 0.6 kV cm–1 for 30 µs. Further studies need to be performed to improve cleavage rates and assess development to the blastocyst stage.

81 COMPARISON OF DEVELOPMENTAL ABILITY AMONG CLONED EMBRYOS WITH VARIOUS INDIVIDUAL RECIPIENT CYTOPLASMS IN BOVINE

2006 ◽  
Vol 18 (2) ◽  
pp. 148
Author(s):  
J. G. Zhao ◽  
X. Y. Yang ◽  
H. F. Liu ◽  
H. Li ◽  
S. Z. Huang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Vero Cells ◽  
Fusion Rate ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Chi Square ◽  
Reconstructed Embryos ◽  
Maturation Rate ◽  
Chi Square Test

Faithful reprogramming ensures the proper activation of genes during embryonic development of the somatic cell nuclear transfer (NT) in bovine. It is unambiguous that all these remodeling factors are presented in the oocyte cytoplasm (Du et al. 2002 Mol. Reprod. Dev. 63, 183–191). It will be interesting to determine if the recipient cytoplasms derived from individuals have different development ability and reprogramming competence during NT. Oocytes recovered by Ovum pickup from five Holstein heifers at 14 months of age were used as recipient cytoplasms. Cultured granulosa cells of the same origin were used as donor cells. Oocytes were enucleated at 20 h post-maturation and a single donor cell was transferred into the perivitelline space of a recipient oocyte. After fusion and activation, the reconstructed embryos were cultured in B2 medium (Laboratoire CCD, Paris, France) on a monolayer of Vero cells for 7 days. The oocyte number, development ability, and NT efficiency of recipient cytoplasm derived from each individual were compared (Table 1). Differences among individuals were verified using a chi-square test, SAS 6.12 version (SAS Institute, Cary, NC, USA). There were significant differences of survival after fusion and the rate of development to the blastocyst stage for embryos reconstructed with recipient cytoplasm from five different individual heifers (P < 0.05). However, maturation rate, fusion rate and cleavage rate of embryos reconstructed with recipient cytoplasm from five different individual heifers presented no significant differences (P > 0.05). Reconstructed embryos with recipient cytoplasm from one heifer (03025) showed a lower survival after fusion (61% vs. 80%, 86%, 77%, 91%) but a higher ability to develop to blastocyst stage (61% vs. 24%, 31%, 52%, 31%) than the embryos from the other four heifers. The current study showed that recipient cytoplasm from various individuals may present great differences in developmental ability in nuclear transfer. This may result from different compatibility between nucleus and mitochondria or the content of maternal RNA as well as proteins in the oocyte. Further studies are needed to elucidate the genetic factors that affect the reprogramming in nuclear transfer. Table 1. Nuclear transfer efficiency with various individual recipient cytoplasms

160 In vitro maturation of pre-pubertal goat oocytes and their development after chemical activation

2019 ◽  
Vol 31 (1) ◽  
pp. 205
Author(s):  
N. A. Wani ◽  
S.-B. Hong
Keyword(s):  
In Vitro Maturation ◽  
Culture Media ◽  
Chemical Activation ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
Epidermal Growth ◽  
Humidified Air ◽  
Activation Status

Experiments were conducted to study in vitro maturation of pre-pubertal goat oocytes and their developmental potential after chemical activation. Cumulus-oocyte complexes (n=1170) collected from the ovaries of pre-pubertal goats slaughtered at a local abattoir were matured in TCM-199 supplemented with 0.15mg mL−1 l-glutamine, 0.25mM sodium pyruvate, 0.1mM l-cysteine, 20ng mL−1 epidermal growth factor, 10mg mL−1 FSH, 10mg mL−1 LH, 1μg mL−1 oestradiol and 10% FCS for 24h at 39°C under 5% CO2 in humidified air. In Experiment 1, matured oocytes were activated (r=6) with either 5mM ionomycin (n=85) or 7% ethanol (n=91) followed by culture in 6-DMAP for 4h. All the activated oocytes were then cultured in KSOM supplemented with 3mg mL−1 BSA and were fixed and stained with Hoechst 33342 after 18h of culture to evaluate their activation status. In Experiment 2, oocytes activated with 5mM ionomycin and 6-DMAP were cultured for 7 days (r=6) in 1 of the 4 different culture media [Charles Rosenkrans medium (CR-1), modified TCM-199, KSOM and SOF] to study their developmental potential. All media were supplemented with 3.0mg mL−1 BSA for the first 3 days and 10% FCS for the subsequent 4 days. Of these pre-pubertal oocytes, 59% reached metaphase II stage, and 83% of these oocytes were classified as activated in the group using ionomycin in comparison with 69% in the group using ethanol as an activating agent (P&lt;0.05). No difference was observed in the cleavage rate of activated oocytes cultured in any of the 4 culture media (65.7v. 55.0v. 61.0v. 56.2%, respectively). However, the development to blastocyst stage was observed in only KSOM (16%) and SOF (5%) media. In conclusion, the present study demonstrates that pre-pubertal goat oocytes can mature in vitro and can be activated with 5mM ionomycin, and KSOM, and to a lesser extent SOF, supports development to the blastocyst stage. We plan to use these oocytes as a cytoplast source for interspecies somatic cell NT; however, before that, more studies are needed to evaluate their requirements in culture media to enhance their development to the blastocyst stage.

Nuclear transplantation using bovine primordial germ cells from male fetuses

1995 ◽  
Vol 7 (5) ◽  
pp. 1217 ◽  
Author(s):  
F Delhaise ◽  
FJ Ectors ◽  
Roover R de ◽  
F Ectors ◽  
F Dessy
Keyword(s):  
Nuclear Transfer ◽  
Primordial Germ Cells ◽  
Blastocyst Stage ◽  
Cell Counting ◽  
Nuclear Transplantation ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Developmental Potential ◽  
Morula Stage

The developmental potential of nuclei of bovine gonial cells was investigated by nuclear transfer. Gonial cells were collected from male fetuses at about 175 days post coitum (p.c.). They were fused with enucleated oocytes; reconstituted embryos were cultured in vitro for 7 days. Embryos reaching the compacted morula or blastocyst stage were either fixed for cell counting or transferred into recipients. Out of 115 oocyte-gonia fusions, 101 (87.8%) gave rise to cleaved embryos at Day 3 and 26 (22.6%) had reached the 8-cell stage. At Day 7, 1 (1%) developed to the morula stage and 5 (4%) reached the blastocyst stage. Three blastocysts were fixed and showed normal cell numbers (135; 90; 76 cells). Three blastocysts and one morula were transferred in four recipients; two recipients were pregnant at Day 21 but only one was positive at Day 35 p.c.; this last one aborted around Day 40 p.c. No conceptus was collected. These results indicate that gonial cell nuclei can be partially reprogrammed; they are able to develop into blastocysts and to initiate gestation. However, more experiments will be necessary to prove the nuclear totipotency of bovine gonial cells.

In vitro production of cattle-water buffalo (Bos taurus - Bubalus bubalis) hybrid embryos

Zygote ◽  
2002 ◽  
Vol 10 (2) ◽  
pp. 155-162 ◽  
Author(s):  
H.P.S. Kochhar ◽  
K.B.C. Appa Rao ◽  
A.M. Luciano ◽  
S.M. Totey ◽  
F. Gandolfi ◽  
...  
Keyword(s):  
Water Buffalo ◽  
Bos Taurus ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bubalus Bubalis ◽  
Blastocyst Stage ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Developmental Potential

Interspecific hybrid embryos are useful models for the study of maternal-fetal interactions, transmission pattern of species-specific markers and parental contributions to growth and developmental potential of pre-attachment embryos. In an attempt to investigate the possibility of producing hybrid embryos of domestic cattle (Bos taurus) and water buffalo (Bubalus bubalis), cattle oocytes were exposed to buffalo sperm and buffalo oocytes were exposed to cattle sperm and the cleavage rate and the post-fertilisation features of hybrid embryos up to the blastocyst stage were compared with those of buffalo and cattle embryos. The cleavage rate in buffalo oocytes exposed to cattle sperm was low (40.8%), with only 8.8% of these hybrid embryos reaching the blastocyst stage. Cattle oocytes exposed to buffalo sperm showed 86.3% cleavage, while 25.9% of these attained the blastocyst stage. The speed of development of both types of hybrids was intermediate between that of cattle and buffalo embryos, with hatching occurring on day 7.5 in hybrid embryos, day 8-9 in cattle and day 7 in buffalo. The proportions of cells contributing to the trophectoderm and the inner cell mass were closer to those of the maternal species in both types of hybrid embryos. Our results indicate that cattle-water buffalo hybrid embryos produced using interspecies gametes are capable of developing to advanced blastocyst stages and that their in vitro fate, and developmental potential, are influenced by the origin of the oocyte.

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