32 A CLONED FOAL PRODUCED USING OOCYTES RECOVERED BY TRANSVAGINAL ASPIRATION OF IMMATURE FOLLICLES

2011 ◽  
Vol 23 (1) ◽  
pp. 122
Author(s):  
Y. H. Choi ◽  
J. D. Norris ◽  
I. C. Velez ◽  
C. C. Jacobson ◽  
D. L. Hartman ◽  
...  
Keyword(s):  
Medical Care ◽  
Histone Deacetylase ◽  
Nuclear Transfer ◽  
Live Birth Rate ◽  
Control Treatment ◽  
Blastocyst Development ◽  
Immature Oocytes ◽  
Sperm Extract ◽  
Transfer Procedures

Closure of all the horse slaughterhouses in the US has reduced the availability of equine oocytes in this country. We investigated the use of oocytes collected from immature follicles of live mares for cloning research. Because blastocyst development of equine cloned embryos is typically low (<10%), we also investigated the effect of Scriptaid, a histone deacetylase inhibitor that increases blastocyst development, live birth rate, and neonatal health in cloned mice and pigs. Immature oocytes were transvaginally aspirated from all follicles ≥8 mm diameter in a herd of 11 mares. The oocytes were cultured in modified M199 for 24 to 26 h. Donor fibroblasts from a 27-year old stallion were treated with roscovitine for 24 h, then were directly injected into enucleated oocytes using the Piezo drill. Reconstructed oocytes were activated with ionomycin followed by injection of sperm extract and culture with 6- dimethylaminopurine (6-DMAP) for 4 h. Recombined oocytes in the Scriptaid treatment were cultured in the presence of Scriptaid, 250 nM, starting at the onset of 6-DMAP treatment and continuing for a total of 18 to 20 h. After embryo culture, blastocysts were shipped for transfer to recipient mares. Overall, each oocyte donor mare underwent aspiration up to 10 times; 653 follicles were aspirated and 271 oocytes were recovered. The in vitro maturation rate was 65% (172/263). After nuclear transfer procedures, 147 oocytes survived; 130 were used for the study. The blastocyst development rate was 2/47 (4%) in the control treatment and 1/83 (1%) in the Scriptaid treatment. All 3 blastocysts yielded pregnancies after transfer. Both control pregnancies were lost, 1 at 30 days and other at 9 months. The mare pregnant with the embryo from the Scriptaid treatment foaled at 326 days of gestation. The foal had medical issues at birth similar to those seen in some cloned foals previously, including maladjustment, patent urachus, and poor oxygenation. These issues were resolved with medical care; the foal is 3 months of age and healthy at the time of writing. These results indicate that immature oocytes obtained from a limited number of mares can be used successfully for nuclear transfer, providing the opportunity to control the mitochondrial identity of the host cytoplast. Scriptaid treatment did not improve the rate of blastocyst development or prevent health problems at birth; however, transfer of 1 embryo in this treatment produced a viable foal. More work is needed to determine the effect of histone deacetylase treatment on efficiency of cloning in the horse. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts. We thank Drs. Malgorzata Pozor, Margo Macpherson, and the Medicine team at the University of Florida for medical care of the foal.

27 EFFECT OF ACTIVATION METHODS AND BIOPSY SAMPLING ON NUCLEAR TRANSFER BLASTOCYST PRODUCTION AND CLONING EFFICIENCY IN A HORSE

2008 ◽  
Vol 20 (1) ◽  
pp. 94
Author(s):  
Y. H. Choi ◽  
D. L. Hartman ◽  
R. A. Fissore ◽  
S. J. Bedford ◽  
K. Hinrichs
Keyword(s):  
Skin Biopsy ◽  
Nuclear Transfer ◽  
Control Method ◽  
Tissue Sample ◽  
Injection Pressure ◽  
Biopsy Sample ◽  
Control Treatment ◽  
Cloning Efficiency ◽  
Blastocyst Development ◽  
Sperm Extract

In the horse, rates of blastocyst production after nuclear transfer are low. This study was conducted to examine the effect of activation via injection of different volumes of sperm extract or via injection of murine mRNA for PLC-ζ, a sperm-specific protein which induces Ca2+ oscillations in all species thus far studied, on blastocyst development after nuclear transfer. Two biopsy samples from the same horse were also examined for cloning efficiency. Donor fibroblasts cultured from a skin biopsy taken from a 19-year-old mare were treated with 15 µm R-roscovitine for 18 to 24 h before direct injection into enucleated ooplasts. Ionomycin treatment, 5 µm for 4 min, was used in all activation treatments, as the combination of ionomycin with sperm extract provided the highest blastocyst development and foaling rates in our previous report (Hinrichs et al. 2007 Reproduction 134, 319–325). All treatments were followed by incubation in 2 mm 6-dimethylaminopurine for 4 h. Differences in blastocyst development between treatments were analyzed using Fisher's exact test. In Experiment 1, reconstructed oocytes were injected with sperm extract for 0.1, 0.2, 0.4, 0.8, or 1.6 s at a fixed pipette diameter and injection pressure, and then treated with ionomycin. The blastocyst rate (9.8%) for 0.1 s was significantly higher than that for 0.2 s (0%) or 0.8 s (1.4%). In Experiment 2, murine PLC-ζ mRNA (0.25 µg µL–1) was injected into reconstructed oocytes 20 to 30 min before or after ionomycin treatment and compared with a control treatment (injection of 2 to 4 pL sperm extract 20 to 50 min after ionomycin exposure). There were no differences in blastocyst development among treatments (0, 4.5, and 5.6%, respectively). Transfer of 10 blastocysts produced in Experiments 1 and 2 resulted in 5 pregnancies: however, all were lost before 70 days of gestation. In Experiment 3, a second skin biopsy was obtained from the same mare and cells from this tissue sample were used for nuclear transfer concurrently with cells from the first sample, using the control method above. Blastocyst production was higher using cells from the second biopsy sample (4/23 v. 0/23; P = 0.05). Transfer of these four blastocysts yielded four pregnancies, two of which continued to term and produced viable foals. These results indicate that blastocyst development after injection of sperm extract is dependent upon the volume injected, that injection of murine PLC-ζ mRNA does not improve blastocyst formation under the given conditions, and that the efficiency of cloning may vary with biopsy sample even from the same animal.

P–804 Morphokinetic development by time-lapse imaging of conceptuses generated from artificial oocytes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A Trout ◽  
P Xie ◽  
A Petrini ◽  
Z Rosenwaks ◽  
G Palermo
Keyword(s):  
Somatic Cell ◽  
Histone Deacetylase ◽  
Nuclear Transfer ◽  
Cumulus Cells ◽  
Time Lapse ◽  
Culture Conditions ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Blastocyst Development ◽  
Time Lapse Imaging

Abstract Study question What are the ideal culture conditions to enhance full preimplantation development of embryos generated by FVB somatic cell haploidization (SCH) in the mouse model? Summary answer The presence of a histone deacetylase inhibitor yielded the best morphokinetic development of expanded blastocysts generated by FVB SCH, comparable to control blastocysts. What is known already Various culture conditions and medium supplements have been proposed to promote preimplantation development of embryos generated by SCH, including supplementation with trichostatin A (TSA), fasudil, scriptaid, and RAD–51 stimulatory compound–1 (RS–1). TSA and scriptaid, both histone-deacetylase inhibitors, have been found to improve embryo development following nuclear transfer by enhancing histone acetylation and cellular reprogramming. Additionally, fasudil is a Rho-associated kinase inhibitor that has been shown to reduce apoptosis and promote cell proliferation. Finally, RS–1 stimulates RAD51 activity, which promotes the repair of DNA damage and increases the efficacy of somatic cell reprogramming. Study design, size, duration B6D2F1 mouse metaphase II (MII) oocytes underwent enucleation and nuclear transfer, or were ICSI inseminated serving as controls. Reconstituted oocytes showing development of a meiotic-like spindle demonstrated successful SCH, and were ICSI inseminated. SCH conceptuses were cultured in one of three groups: KSOM, KSOM supplemented with TSA (TSA), or KSOM supplemented with fasudil, scriptaid, and RS–1 (Cocktail). ICSI controls (ICSIC) were cultured in KSOM medium. Fertilization and full preimplantation development were compared among all groups. Participants/materials, setting, methods Ooplasts were generated from MII oocytes by removing spindle complexes under OosightÔ visualization and cytochalasin B exposure. A single FVB mouse cumulus cell was transferred into the perivitelline space and fused with the ooplast, facilitated by Sendai virus. Reconstructed oocytes with novel pseudo-meiotic spindles underwent piezo-ICSI and were cultured in different media conditions in a time-lapse imaging system up to 96h. TSA and Cocktail embryos had media changed to regular KSOM 10 hours after insemination. Main results and the role of chance A total of 274 B6D2F1 MII oocytes were enucleated, resulting in a 95.9% survival rate. All ooplasts survived nuclear transfer and 62.1% successfully haploidized after 2 hours. ICSIC and reconstituted SCH oocytes survived piezo-ICSI at rates of 81.5% and 57.0%, respectively (P &lt; 0.01). SCH embryos were then allocated into KSOM, TSA supplied, and Cocktail media. Fertilization rates for ICSIC, KSOM, and TSA embryos were 92.4%, 90.7%, and 94.4%, respectively, while the rate for embryos cultured in Cocktail was only 71.9% (P &lt; 0.03). While embryos cultured in Cocktail had a comparable 2-cell timing to ICSIC, embryos in TSA reached developmental milestones with a closer timing to the ICSIC, having minor delays at the 3-, 4-, and 6-cell stages (P &lt; 0.05). KSOM- and Cocktail-cultured embryos were delayed at most of the stages (P &lt; 0.01), except for the two-pronuclei appearance. Although the TSA group displayed the best embryo developmental pattern, the final rate of blastocyst development was somewhat homogeneous with rates of 15.4%, 23.5%, and 13.0% for the KSOM, TSA, and Cocktail groups, respectively (P &lt; 0.001), and remarkably lower than the ICSIC (81.6%). Limitations, reasons for caution Although live pups have been obtained using BDF cumulus cells, embryos generated by FVB cumulus cells show a remarkably lower blastocyst development, but maintain morphokinetic characteristics similar to ICSIC in the presence of TSA. Wider implications of the findings: While using different strains to enhance genetic variance, the morphokinetic analysis of preimplantation embryos in ideal culture conditions is paramount to the progress of neogametogenesis. The implementation of this technique may soon help create genotyped oocytes for women with compromised ovarian reserve. Trial registration number N/A

30 NUCLEAR AND MICROTUBULE DYNAMICS IN SOMATIC CELL NUCLEAR TRANSFER SHEEP EMBRYOS ACTIVATED WITH T-DIMETHYLAMINOPURINE AND CYCLOHEXIMIDE

2006 ◽  
Vol 18 (2) ◽  
pp. 123
Author(s):  
G. Coppola ◽  
B.-G. Jeon ◽  
B. Alexander ◽  
E. St. John ◽  
D. H. Betts ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Laser Scanning ◽  
Cell Cycle Progression ◽  
Treated Group ◽  
Blastocyst Development ◽  
Fetal Fibroblasts

The early reprogramming events following somatic cell nuclear transfer (SCNT) determine the fate of the cloned embryo and its development to a healthy viable offspring. In the present study, we undertook a detailed immunocytochemical study of the patterns of both microtubules and chromatin during the first cell cycle of sheep nuclear transfer embryos after fusion and artificial activation using either 6-dimethylaminopurine (6-DMAP) or cycloheximede (CHX). Sheep oocytes were collected from abattoir ovaries and matured in vitro for 18-20 h and enucleated; fetal fibroblasts were transplanted using standard SCNT techniques. Reconstructed cell-cytoplast couplets were fused and activated with ionomycin, followed by culture in two separate groups containing 6-DMAP (2 mM) or CHX (10 �g/mL) for 3 h. Following activation, embryos were cultured in in vitro culture (IVC) medium for blastocyst development. Embryos (n = 15, 3 replicates) were randomly removed from culture at various time points and stained using standard immunocytochemical methods to observe microtubule and nuclear configurations. Images were captured using laser scanning confocal microscopy. Results reveled that at 1 h post-fusion, 63.3% of reconstructed embryos underwent nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) was apparent as chromosomes were situated on a non-polar spindle. The remaining embryos showed abnormal spindle and DNA configurations including chromosome outliers, congression failure, and non-NEBD. At 1 h post-activation (hpa), the embryos treated with 6-DMAP had already formed a clearly visible pronucleus (diameter 6-8 �m), whereas in the CHX-treated group, none of the embryos were at pronuclear stage; instead most of the latter embryos showed two masses of chromatin. At 1 hpa, 6-DMAP- and CHX-treated embryos showed one swelled pronucleus with a mean diameter of 8.4 � 1.3 �m and 25.8 � 0.8 �m, respectively (P < 0.05). At 16 hpa, embryos from both treatment groups still showed one swelled pronucleus. In the 6-DMAP-treated embryos, most of the embryos showed a metaphase spindle with aligned chromosomes of the first mitotic division as early as 18-10 hpa, whereas in the CHX-treated group embryos were still at the pronuclear stage. Typical 2-cell division was seen in most of the 6-DMAP-treated embryos between 24 and 30 hpa, but it was slightly delayed in CHX-treated embryos (32-35 hpa). Blastocyst development rates in the 6-DMAP- and CHX-treated groups were 21.4 � 5.6% and 14.0 � 6.3%, respectively (P < 0.05). In summary, artificial activating agents 6-DMAP and CHX exhibited different effects on chromatin remodeling, cell cycle progression, and the degree of pronuclear swelling which may explain the poor developmental rates and abnormal chromosome complements observed for cloned embryos. This work was funded by NSERC, OMAF, and International Council for Canadian Studies.

38 EFFECT OF OOCYTE MATURATION DURATION ON BLASTOCYST RATES AFTER EQUINE SOMATIC CELL NUCLEAR TRANSFER

2015 ◽  
Vol 27 (1) ◽  
pp. 112 ◽  
Author(s):  
Y. H. Choi ◽  
I. C. Velez ◽  
B. Macías-García ◽  
K. Hinrichs
Keyword(s):  
Somatic Cell ◽  
Oocyte Maturation ◽  
Nuclear Transfer ◽  
Transvaginal Ultrasound ◽  
Direct Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Blastocyst Development ◽  
Donor Cells

In equine cloning, the scarcity of equine oocytes places emphasis on development of the most efficient nuclear transfer (NT) methods possible. In other species, using oocytes matured for the shortest duration needed to reach metaphase II has increased NT efficiency. In the present study, we examined the effect of duration of oocyte maturation at the time of enucleation on equine cloned blastocyst production. Oocytes were collected from live mares by transvaginal ultrasound-guided aspiration of all visible follicles ≥5 mm in diameter. The oocytes were held overnight (16–22 h) at room temperature, matured in vitro, and reconstructed with donor cells as described in our previous study (Choi et al. 2013 Theriogenology 79, 791–796). In Experiment 1, oocytes were divided into 2 groups and matured for 20 or 24 h. After enucleation, oocytes were reconstructed by direct injection of donor cells. Reconstructed oocytes were held for 5 h and then activated by treatment with 5 μM ionomycin for 4 min, then injection with sperm extract, followed by incubation in 2 mM 6-DMAP for 4 h. The activated reconstructed oocytes were cultured in global human embryo culture medium under 5% CO2, 6% O2, and 89% N2 at 38.2°C for 7 to 11 days (20 mM glucose was added at Day 5) and blastocyst rate was recorded. Because a low maturation rate was found at 20 h in Experiment 1, in Experiment 2 oocytes were denuded at 20 h and those that were mature were enucleated and used for NT; those that had not cast out a polar body at 20 h were cultured for an additional 3 h (20 + 3h) and then evaluated for polar body formation and used for NT, which was conducted as in Experiment 1. Data were analysed by Fisher's exact test. In Experiment 1, 203 oocytes were collected in 46 aspiration sessions. The rate of oocyte maturation to metaphase II was significantly lower for oocytes cultured for 20 h (35/116, 30%), than for those cultured for 24 h (47/80, 59%). However, the rate of blastocyst development was significantly higher for oocytes cultured for 20 h (11/27, 41%) than for 24 h (2/38, 5%). In Experiment 2, 89 oocytes were collected in 18 aspiration sessions. After 20 h of maturation culture, 22 oocytes were mature (25%). After an additional 3 h of culture, 21 additional oocytes had matured. There were no significant differences between the two treatments (20 and 20 + 3h) in reconstruction rates (77%, 17/22, and 90%, 19/21, respectively) or blastocyst rates (24%, 4/17, and 32%, 6/19, respectively). These results indicate that duration of in vitro maturation, or the duration of presence of cumulus cells, influences blastocyst development after somatic cell NT in the horse. This appears to be due to a benefit of using oocytes immediately after they reach metaphase II; if this is ensured as in Experiment 2, the duration of maturation itself had no effect.This work was supported by the American Quarter Horse Foundation, the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts.

Somatic cell nuclear transfer using transported in vitro-matured oocytes in cynomolgus monkey

Zygote ◽  
2007 ◽  
Vol 15 (1) ◽  
pp. 25-33 ◽  
Author(s):  
N. Chen ◽  
S-L. Liow ◽  
R. Bin Abdullah ◽  
WK. Khadijah Wan Embong ◽  
W-Y. Yip ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Oocyte Retrieval ◽  
Development Rate ◽  
Cleavage Rate ◽  
Immature Oocytes ◽  
Polarized Microscopy

SUMMARYSomatic cell nuclear transfer (SCNT) is not successful so far in non-human primates. The objective of this study was to investigate the effects of stimulation cycles (first and repeat) on oocyte retrieval and in vitro maturation (IVM) and to evaluate the effects of stimulation cycles and donor cell type (cumulus and fetal skin fibroblasts) on efficiency of SCNT with transported IVM oocytes. In this study, 369 immature oocytes were collected laparoscopically at 24 h following human chorionic gonadotrophin (hCG) treatment from 12 cynomolgus macaque (Macaca fascicularis) in 24 stimulation cycles, and shipped in pre-equilibrated IVM medium for a 5 h journey, placed in a dry portable incubator (37 °C) without CO2 supplement. A total of 70.6% (247/350) of immature oocytes reached metaphase II (MII) stage at 36 h after hCG administration, MII spindle could be seen clearly in 80.6% (104/129) of matured IVM oocytes under polarized microscopy. A total of 50.0% (37/74) of reconstructive SCNT embryos cleaved after activation; after cleavage, 37.8% (14/37) developed to the 8-cell stage and 8.1% (3/37) developed to morula, but unfortunately none developed to the blastocyst stage. Many more oocytes could be retrieved per cycle from monkeys in the first cycle than in repeated cycles (19.1 vs. 11.7, p < 0.05). There were no significant differences in the maturation rate (70.0 vs. 71.4%, p > 0.05) and MII spindle rate under polarized microscopy (76.4 vs. 86.0%, p > 0.05) between the first and repeat cycles. There were also no significant differences in the cleavage rate, and the 4-cell, 8-cell and morula development rate of SCNT embryos between the first and repeat cycles. When fibroblast cells and cumulus cells were used as the donor cells for SCNT, first cleavage rate was not significantly different, but 4-cell (50.0 vs. 88.9%, p < 0.05) and 8-cell (0 vs. 51.9%, p < 0.01) development rate were significantly lower for the former. In conclusion, the number of stimulation cycles has a significant effect on oocyte retrieval, but has no effect on maturation and SCNT embryo development; however, different donor cell types (cumulus and fibroblast) resulted in different developmental potentials of SCNT embryos.

scholarly journals Induced parturition in recipient cattle carrying nuclear transfer calves

2002 ◽  
Vol 45 (5) ◽  
pp. 443-449
Author(s):  
D. J. Funk
Keyword(s):  
Medical Care ◽  
Pulmonary Edema ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Health Problems ◽  
Caesarian Section ◽  
Postnatal Period ◽  
Placental Pathology ◽  
Placental Dysfunction

Abstract. Historically there has been a lack of spontaneous parturition in nuclear transfer animals produced from somatic cells. A significant number of nuclear transfer calves are born with health problems and died within a week of birth. To a lesser degree in-vitro produced (IVP) calves have had the same abnormalities at birth. There are numerous reports of nuclear transfer calves having placental dysfunction such as hydroallantois or hydroamnion, and placental pathology such as edema or extremely large and/or abnormal placentomes. In an attempt to circumvent some of these maladies, a program was devised to induce recipients carrying nuclear transfer fetuses to calve 1 week earlier than expected full term gestation. All recipient cows were induced toward parturition at day 274 (+/- 2 days) of gestation. The cows were injected IM with 20 mg of dexamethasone (Pro Labs Ltd., St. Joseph, MO, U.S.A.) and 25 mg dinoprost (Pharmacia &amp; Upjohn, Kalamazoo, MI, U.S.A.) 24 hours before an elective caesarian section. Of the 31 nuclear transfer calves born at Trans-Ova Genetics utilizing this procedure, 23 (74%) survived the immediate postnatal period with minimal medical care. Eight calves (26%) died within 36 hours of birth. Placental edema, large birth weights, pulmonary edema and pathological right side heart problems were common findings in the calves that died. Two calves (6%) died later in life from other unrelated health problems. 21 calves (68%) remain for productive ventures. Birth weights on all calves ranged from 38 to 89 kg.

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).

58 ISOLATION OF BOVINE TROPHOBLAST AND ITS REPROGRAMMING BY NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 134
Author(s):  
I. M. Saadeldin ◽  
B. H. Kim ◽  
B. Roibas da Torre ◽  
O. J. Koo ◽  
G. Jang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Quantitative Polymerase Chain Reaction ◽  
Cell Mass ◽  
Donor Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Embryonic Cells ◽  
Blastocyst Development

Nuclear transfer (NT) has been used to produce many cloned offspring using several types of cells, including embryonic cells. Even though inner cell mass cells have been used as donor karyoplast for producing cloned animals, there are few studies using trophoblast. In mice, clones were born by nuclear transfer of trophoblasts from the expanded blastocyst into enucleated oocytes as a trial to show the totipotency of both inner cell mass and trophectoderm cells isolated from blastocysts (Tsunoda and Kato 1998 J. Reprod. Fertil. 113, 181–184). However, bovine trophoblast cell (TC) lines have not been used in NT to date. The purpose of this study was to elucidate whether TC as donor cell can be reprogrammed in bovine enucleated oocyte and determine the relative abundance of interferon tau (IFNτ) expression in the resulting cloned preimplantational embryos. Hatched blastocysts produced by IVF were used to isolate TCs on mouse embryonic fibroblasts treated with mitomycin C as feeder cells. TCs and adult fibroblasts (AF, control group for NT) were microinjected to perivitelline space of in vitro mature enucleated oocytes and electrically fused. Reconstructed embryos were chemically activated and cultured in a 2-step chemically defined medium. Levels of IFNτ expression in IVF-, TC-, and AF-derived blastocysts were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). IVF produced embryos were used as reference to analyze the linear progressive expression of IFNτ through mid-, expanded, and hatching blastocysts. As a result, TCs expressing IFNτ were successfully isolated and cultured on feeder layers. It grew as cell sheets of cuboidal epithelium with high proliferation capacity as a single colony originated from a small clump of cells measured 0.5 cm within 7 days of culture. TCs were reprogrammed in the enucleated oocytes to blastocyst with similar efficiency to AF (14.5% and 15.6%, respectively; P ≤ 0.05). RT-qPCR studies showed that IFNτ expression was higher in TC-derived blastocysts than IVF- and AF-derived blastocysts. Both IVF- and TC-derived blastocysts, showed progressive increase of IFNτ expression through the advancement of blastocyst development when it was compared to AF-derived blastocysts. In conclusion, using TCs expressing IFNτ as donor cell for bovine NT could increase the developmental competence of cloned embryos as indicated by progressive linear increase in IFNτ expression. This study was supported by grants from IPET (#109023-05-1-CG000), NRF (#M10625030005-10N250300510), MKE (#2009-67-10033839, #2009-67-10033805), and BK21 program. Saadeldin I. M. is supported by Islamic Development Bank (IDB) merit scholarship, Jeddah, Saudi Arabia.

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).

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