81 MOUSE CLONING BY USING A LASER-ASSISTED ZONA OPENING AND ELECTRO-FUSION TECHNIQUE

2007 ◽  
Vol 19 (1) ◽  
pp. 158
Author(s):  
R. Rungsiwiwut ◽  
A. Thongphakdee ◽  
P. Numchaisrika ◽  
P. Virutamasen ◽  
M. Techakumphu
Keyword(s):  
Zona Pellucida ◽  
Polar Body ◽  
National Research Council ◽  
Donor Cell ◽  
Developmental Rate ◽  
Fusion Rate ◽  
Cleavage Rate ◽  
Fusion Technique ◽  
First Polar Body ◽  
Chi Square Analysis

Mouse cloning can be performed by a direct microinjection of donor nuclei using a conventional or a piezo-actuated technique (Rybouchkin et al. 2002 Reproduction 124, 197–207; Wakayama et al. 1998 Nature 394, 369–374). However, a high percentage of lysed oocytes was observed during the pipette penetration of the cytoplasmic membrane through the zona pellucida. The aim of this experiment was to investigate the possibility of a combination of a laser-assisted zona opening and electro-fusion for mouse cloning. Mature oocytes were obtained from FSH-superovulated B6D2F1 female mice. Enucleation and transfer of donor cell were performed in HEPES-buffered CZB medium. Spindle-chromosome complexes (SCCs) together with first polar body were removed by blunt-end pipette via a small hole in the zona pellucida which was cut by a laser beam. An adult fibroblast cell was introduced into the perivitelline space and fused to the enucleated oocyte by using a single DC pulse of 1.5 kV cm-1, 20 �s, in a fusion medium (Liu and Aoki 2003 Animal Sci. J. 75, 125–129). The fusion rate was checked 30 min later and only the fused oocytes were subjected to activation by 6 h culture in Ca2+-free CZB medium supplemented with 10 mM Sr2+ and 5 �g mL-1 cytochalasin B. The oocytes which presented the pseudo-pronuclei were considered as the activated oocytes and were cultured in CZB medium at 37�C, 5% CO2 in humidified atmosphere. The developmental rate was observed every 24 h for 4 days. The diploid parthenogenetically activated embryos serving as a control were obtained using the same activation protocol but without SCC removal. The percentages of survival after enucleation and after fusion were recorded. The formation of pseudo-pronuclei and the embryos developing to a particular stage were determined by chi-square analysis. The results show that most of the oocytes survived after enucleation (92.5%, 172/186) and the fusion rate was 71.9% (105/146). The formation of pseudo-pronuclei and the cleavage rate of cloned embryos was lower than in the control (87.6% (92/105) vs. 100% (90/90) and 69.6% (64/92) vs. 92.2% (83/90), respectively). The developmental rate to morula–blastocyst stage of cloned embryos was significantly lower than in the control [1.1% (1/92) vs. 44.4% (40/90); P < 0.05]. These results indicate that using laser-assisted zona opening and electro-fusion technique is practical for mouse cloning and provides an alternative method when injection of donor nuclei into the recipient oocytes using a conventional or a piezo-driven method is technically difficult. This study was supported by grants from The National Research Council of Thailand and The Thailand Research Fund (Loyal Golden Julilee Ph.D. program).

81 THE EFFECT OF FOLLICULAR AND OVIDUCT OOCYTES ON THE DEVELOPMENT OF RABBIT NUCLEAR TRANSFERRED EMBRYOS IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 199
Author(s):  
L.-Y. Sung ◽  
C.-H. Chen ◽  
T.-A. Lin ◽  
L.-J. Sung ◽  
H.-Y. Su ◽  
...  
Keyword(s):  
Polar Body ◽  
Fluorescent Microscopy ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Fusion Rate ◽  
Adult Rabbit ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
First Polar Body

This study was designed to examine the effect of rabbit oocytes collected from oviducts v. follicles on the developmental potential of nuclear transplant (NT) embryos. Rabbit oocytes were flushed from the oviducts (oviduct oocytes) or collected from the ovarian Graafian follicles(follicular oocytes) of superovulated does at 12 h post-hCG injection (hpi). Cumulus cells were then removed from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in TCM-199 +10% fetal bovine serum (FBS) and confirmed under fluorescent microscopy. Skin fibroblasts from an adult rabbit were prepared and cultured to passage 8 to 10 before use as nuclear donors. A donor cell with a diameter of approximately 15 to 19 μm was transferred into the perivitelline space of an enucleated oocyte and subsequently fused with the recipient oocyte by applying 3 direct current pulses at 3.2 kV cm-1 for 20 μs per pulse. Fused oocytes were activated by the same electrical stimulation described above, and then cultured in TCM-199 + 10% FBS containing 2.0 mM 6-DMAP and 5 μg mL-1 cycloheximide for 1 h. Cloned embryos were cultured in 2.5% FBS B2 medium in 5% CO2 and 95% humidified air at 38.5°C for 3 d. Embryo development to cleavage (2- to 4-cell), 8-cell, and morula/blastocyst (Mor/BL) stages was evaluated. The data were analyzed by the General Linear Model procedure (SPSS 11.0, SPSS Inc., Chicago, IL, USA).The total number of oocytes collected per animal was 27.6 ± 1.3, with 47.8% from oviducts, and 52.2% from follicles. The percentage of oviduct oocytes that showed the first polar body was 98.3% (n = 150) at the time of collection, whereas follicular oocytes only had 54.8% at collection (n = 93), but it reached 92.4% when immature follicular oocytes were cultured for 3 h in vitro. The enucleation rates were similar between the follicular (82.7%) and the oviduct (79.1%) groups. Table 1 shows that a significantly higher fusion rate was found in follicular oocytes compared with that in the oviduct group (90.8 v. 63.4%; P < 0.05). There was no difference in the cleavage rate and Mor/BL development between the 2 groups, although the 8-cell(78.4 v. 63.9%; P = 0.11) and the overall efficiencies (30.6% v. 17.9%; P = 0.14) appeared higher in the follicular group. These results demonstrated that rabbit follicular oocytes at 12 hpi have potential equivalent or maybe better (fusion) than that with oviduct oocytes for promoting the preimplantational development of NT embryos. Table 1.The effect of follicular and oviduct oocytes on the development of rabbit NT embryos Supported by NIH1R43 RR023774-01A1 and 5R44HL091605-03.

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.

scholarly journals 59 A PRELIMINARY STUDY OF THE IN VITRO DEVELOPMENT OF ASIAN ELEPHANT, CLONED EMBRYOS, RECONSTRUCTED USING A RABBIT RECIPIENT OOCYTE

2005 ◽  
Vol 17 (2) ◽  
pp. 179 ◽  
Author(s):  
P. Numchaisrika ◽  
R. Rungsiwiwut ◽  
A. Thongpakdee ◽  
M. Techakumphu
Keyword(s):  
Polar Body ◽  
Calf Serum ◽  
Developmental Rate ◽  
Asian Elephant ◽  
Donor Cells ◽  
First Polar Body ◽  
Electrical Pulses ◽  
Chi Square Analysis

Interspecies nuclear transfer is an important tool for studying the interaction between the cytoplasm of one cell and the donor nucleus of another (Chen et al. 2002 Biol. Reprod. 67, 637–642). The aim of this experiment was to investigate the possibility of developing in vitro an asian elephant cloned embryo using a rabbit recipient oocyte. The elephant donor cells were obtained from the ear skin of a stillborn Asian elephant (Elephus maximus) and the in vivo-matured recipient oocytes were obtained from FSH-stimulated New Zealand White doe rabbits. Enucleation was accomplished by aspiration of the first polar body and the metaphase II plate together with a small amount of cytoplasm. Successful enucleation was confirmed by UV examination after staining with 5 μg mL−1 Hoechst 33342. The donor cells were introduced into the perivitelline space of the enucleated oocytes immediately after enucleation. The elephant-rabbit reconstructed embryos were fused in 0.3 M manitol with 0.1 mM Ca2+ and Mg2+ using two types of electrical pulses: E1 (n = 61): 3.2 kV/cm, 3 pulses, 20 μs (Chesne et al. 2002 Nat. Biotechnol. 20, 366–369); E2 (n = 69): 2.0 kV/cm, 2 pulses, 20 μs (Chen et al. 2002 Biol. Reprod. 67, 637–642). The fused embryos were activated 1 h after fusion by electrical pulses to those used for fusion and then incubated in 5 μg mL−1 cyclohexamide and 2 mM 6-DMAP for 1 h. Subsequently, the activated embryos were cultured in B2 medium containing 2.5% fetal calf serum. The developmental rate was observed every 24 h for 7 days and the differences in the percentages of embryos developing to a particular stage were determined by chi-square analysis. The results showed that the fusion and cleavage rates of elephant-rabbit cloned embryos fused and activated by E1 were significantly higher than for E2 (P < 0.05; see Table 1). Compared with rabbit-rabbit cloned embryos using adult skin fibroblast as a donor cell and E1 for both fusion and electrical activation, we found that the cleavage and blastocyst rates of elephant-rabbit cloned embryos was higher than for the rabbit-rabbit ones (65% (28/43) versus 58% (28/48) and 7% (3/43) versus 4% (2/48) respectively). Results from this study showed that either of the electrical pulses, 3.2 kV/cm, 3 pulses, 20 μs or 2.0 kV/cm, 2 pulses, 20 μs, can be used to fuse elephant somatic cells to rabbit ooplasm and the rabbit oocytes can be served as recipient oocytes to support the development of elephant cloned embryos up to the blastocyst stage. Table 1. Developmental rate of elephant–rabbit cloned embryos after being fused by different electrical pulses This work was supported by Rajadapisek Sompoj Fund, Chulalongkorn University.

111 SLOW FREEZING AND VITRIFICATION OF IN VITRO-MATURED DOMESTIC CAT OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 173 ◽  
Author(s):  
J. Braun ◽  
C. Otzdorff ◽  
T. Tsujioka ◽  
S. Hochi
Keyword(s):  
Polar Body ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Slow Freezing ◽  
Developmental Potential ◽  
Freezing Medium ◽  
First Polar Body ◽  
Blastocyst Rate ◽  
Chi Square Analysis

The effects of slow freezing or vitrification as well as exposure to the cryoprotective media without cooling and warming of in vitro-matured domestic cat oocytes on the in vitro development to the blastocyst stage was investigated. Cumulus–oocyte complexes were matured for 24 h in TCM-199 supplemented with 3 mg mL−1 BSA, 1 µg mL−1 estradiol, 0.1 IU mL−1 FSH, and 0.0063 IU mL−1 LH. Denuded oocytes with a detectable first polar body were inseminated with 2 × 106 cells mL−1 cauda epididymal spermatozoa for 22 h in TALP solution. Presumptive zygotes were cultured in modified SOF medium at 38.5°C in 5% CO2 in air. For slow freezing, oocytes were equilibrated for 20 min at ambient temperatures in PBS with 20% FCS containing either 1.5 M ethylene glycol (EG) + 0.2 M sucrose or 1.5 M EG + 0.2 M trehalose. Oocytes were loaded into 0.25-mL straws, cooled to −7°C at 2°C min, held for 5 min, seeded, cooled down to −30°C at 0.3°C min, and finally plunged into liquid nitrogen. The straws were thawed for 5 s at room temperature and for 30 s in a waterbath at 30°C. Oocytes were washed 3 times before insemination. In vitro-matured oocytes were exposed to the cryoprotective media for 30 min before they were inseminated and then they were cultured for 7 days. For vitrification (Hochi et al. 2004 Theriogenology 61, 267–275), a minimum-volume cooling procedure using Cryotop (Kitazato Supply Co., Tokyo, Japan) as a cryodevice was applied. No blastocysts could be obtained after slow freezing with a cryoprotective medium containing 0.2 M sucrose. Simple exposure to the same freezing medium after in vitro maturation without cryopreservation resulted in a blastocyst rate of 7.9% (control oocytes, 10.7%; not significant (NS); chi-square analysis). Use of trehalose as an extracellular cryoprotectant resulted in the harvest of one blastocyst (0.6%) after slow freezing. Exposure to the same cryoprotective medium resulted in a blastocyst rate of 10.0% (fresh control, 10.9%; NS). After exposure of in vitro-matured oocytes to the vitrification solution, a blastocyst rate of 16.0% was observed (8/50), which was not statistically different from the blastocyst rate in fresh control oocytes (16.3%; 15/92). No blastocysts could be obtained after vitrification (0/64). The results (Table 1) demonstrate that there is no obvious toxic effect of the cryoprotectants employed here for slow freezing or vitrification on the in vitro-matured oocytes, but the developmental potential of cryopreserved oocytes to the blastocyst stage is severely impaired. Table 1. Effect of slow freezing or exposure to freezing medium of matured cat oocytes on the development to the blastocyst stage in vitro

168 miRNA LEVELS DURING BOVINE PREIMPLANTATION EMBRYONIC DEVELOPMENT

2008 ◽  
Vol 20 (1) ◽  
pp. 164
Author(s):  
D. K. Berg ◽  
S. E. Beaumont ◽  
P. L. Pfeffer
Keyword(s):  
Cellular Proliferation ◽  
Expression Profiles ◽  
Polar Body ◽  
Gene Expression Profiles ◽  
Donor Cell ◽  
Vitro Fertilization ◽  
Mrna Targets ◽  
Copy Numbers ◽  
First Polar Body

MicroRNAs (miRNAs) are a class of naturally occurring non-coding RNAs that play a role in gene regulation. They are highly conserved, single-stranded RNAs, 22 nucleotides in length, that are cleaved from larger inactive hairpin precursor transcripts, and use the RNA interference-related pathways to repress their mRNA targets. They play diverse regulatory roles in cellular proliferation, morphogenesis, apoptosis, and differentiation. Maternal miRNAs are crucial for early mammalian development (Murchison et al. 2007 Genes Dev. 21, 682–693; Tang et al. 2007 Genes Dev. 21, 655–648), while sperm-borne miRNAs do not contribute significantly to miRNAs in the zygote (Amanai et al. 2006 Biol. Reprod. 75, 877–884). Our objective was to identify miRNAs that are expressed during bovine in vitro oocyte maturation (MII) and blastocyst stages as well as during parthenogenic development. MII oocytes (n = 1680) were generated from abattoir-derived oocytes and matured in vitro for 24 h. Cumulus cells were removed and the first polar body was visually assessed before the oocytes were frozen in liquid N2. Parthenogenic blastocysts (n = 575) were produced using ionomycin/6DMAP activation, and IVF blastocysts (n = 1150) were produced using standard in vitro fertilization followed by in vitro culture in synthetic oviduct fluid (Thompson et al. 2000 J. Reprod. Fertil. 118, 47–55). Blastocysts (grades 1 and 2) were selected on Day 7 post-activation/insemination and frozen in liquid N2. RNA was isolated using the mirVana miRNA isolation kit (Ambion, Scoresby, Victoria, Australia). miRNAs were quantified using the TaqMan� MicroRNA Human Panel-Early Access Kit (Applied Biosystems, Scoresby, Victoria, Australia) following the manufacturer's protocol. Absolute copy numbers per embryo were estimated. Of the 157 miRNAs in the panel, 102, 136, and 118 were detected above background in oocytes, IVF, and parthenogenic blastocysts, respectively. Only 28 miRNAs were present at over 100 copies in MII oocytes, with maximum levels reaching 1300 copies. Levels were generally much higher at blastocyst stages, with 21 miRNAs present at more than 10 000 copies. miR-16 was one of the most abundant miRNAs in all samples tested. Copy numbers per blastomere cell were 5-fold higher in IVF blastocysts compared to parthegenotic blastocysts for miR-19a, 21, and 30b. The low copy numbers of mature miRNAs before embryonic genome activation may have implications for somatic cell nuclear transfer experiments in that exogenously added miRNAs from the donor cell could impact on the embryonic gene expression profiles.

98 BLASTOCYST DEVELOPMENT RATE OF CLONED CAT EMBRYOS USING SERIAL CLONING

2007 ◽  
Vol 19 (1) ◽  
pp. 166
Author(s):  
X. J. Yin ◽  
H. S. Lee ◽  
E. G. Choi ◽  
X. F. Yu ◽  
B. H. Choi ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Second Generation ◽  
Assisted Reproductive Technologies ◽  
Polar Body ◽  
Donor Cell ◽  
Fibroblast Cell ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Blastocyst Development ◽  
First Polar Body

Domestic cats are a useful research model to develop assisted reproductive technologies for the conservation of endangered felids. Previously, we produced cloned offspring derived from somatic cell nuclear transfer of ear skin fibroblasts obtained from a deaf, odd-eyed, male Turkish Angora. The aim of this study was to assess the cloning efficiency of the fibroblasts derived from a cloned cat. Fibroblast cell lines were established from 6-mm skin biopsies taken from a deaf, odd-eyed, male Turkish Angora and his clone. The protocol for nuclear transfer was described previously (Yin et al. 2005 Reproduction 129, 245–249). Briefly, cumulus cells were removed from the ova by gently pipetting them into TCM-199 supplemented with 0.1% hyaluronidase. The denuded oocytes were then cultured in TCM-199 supplemented with 0.2 �g mL-1 demecolcine for 1 h and placed into TCM-199 containing 5 �g mL-1 cytochalasin B and 0.2 �g mL-1 demecolcine. The first polar body and protruded chromatin plate were removed with a beveled micropipette. Micromanipulation was used to place a single donor cell nucleus into the perivitelline space of enucleated ova. The ovum-cell couplets were fused and pulse activated. The activated couplets were cultured in 500 �L of CRI medium supplemented with 0.3% BSA for 2 days. The cleaved embryos were cultured in CRII medium supplemented with 10% FBS for 5 days. The cleavage and blastocyst development rates were 38.5% and 3.5% for second generation cloned embryos. A total of 310 second generation cloned embryos were transplanted to 9 surrogates, and 2 pregnancies at 30 days were determined by ultrasonography. One pregnancy was aborted at 40 days of gestation; the second pregnancy continued. These results indicate that the serial cloning of a cat can be generated efficiently up until pregnancy. This work was supported by KOSEF (grant #M10525010001-05N2501-00110).

44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS

2011 ◽  
Vol 23 (1) ◽  
pp. 128
Author(s):  
J. Lee ◽  
J. Park ◽  
Y. Chun ◽  
W. Lee ◽  
K. Song
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Skin Fibroblasts ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.

scholarly journals Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

Reproduction ◽  
2002 ◽  
pp. 455-465 ◽  
Author(s):  
YH Choi ◽  
CC Love ◽  
LB Love ◽  
DD Varner ◽  
S Brinsko ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
First Polar Body ◽  
Bovine Oocytes

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.

scholarly journals Grape Seed Proanthocyanidin Ameliorates FB1-Induced Meiotic Defects in Porcine Oocytes

Toxins ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 841
Author(s):  
Wenhui Li ◽  
Yijing He ◽  
Hongyu Zhao ◽  
Lei Peng ◽  
Jia Li ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Polar Body ◽  
Grape Seed ◽  
Ros Generation ◽  
Mrna Levels ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
First Polar Body ◽  
Grape Seed Proanthocyanidin

Fumonisin B1 (FB1), as the most prevalent and toxic fumonisin, poses a health threat to humans and animals. The cytotoxicity of FB1 is closely related to oxidative stress and apoptosis. The purpose of this study is to explore whether Grape seed proanthocyanidin (GSP), a natural antioxidant, could alleviate the meiotic maturation defects of oocytes caused by FB1 exposure. Porcine cumulus oocyte complexes (COCs) were treated with 30 μM FB1 alone or cotreated with 100, 200 and 300 μM GSP during in vitro maturation for 44 h. The results show that 200 μM GSP cotreatment observably ameliorated the toxic effects of FB1 exposure, showing to be promoting first polar body extrusion and improving the subsequent cleavage rate and blastocyst development rate. Moreover, 200 μM GSP cotreatment restored cell cycle progression, reduced the proportion of aberrant spindles, improved actin distribution and protected mitochondrial function in FB1-exposed oocytes. Furthermore, reactive oxygen species (ROS) generation was significantly decreased and the mRNA levels of CAT, SOD2 and GSH-PX were obviously increased in the 200 μM GSP cotreatment group. Notably, the incidence of early apoptosis and autophagy level were also significantly decreased after GSP cotreatment and the mRNA expression levels of BAX, CASPASE3, LC3 and ATG5 were markedly decreased, whereas BCL2 and mTOR were observably increased in the oocytes after GSP cotreatment. Together, these results indicate that GSP could exert significant preventive effects on FB1-induced oocyte defects by ameliorating oxidative stress through repairing mitochondrial dysfunction.

scholarly journals 298RED DEER (CERVUS ELAPHUS) PARTHENOGENETIC BLASTOCYSTS PRODUCED USING IONOMYCIN/6DMAP ACTIVATION

2004 ◽  
Vol 16 (2) ◽  
pp. 268 ◽  
Author(s):  
S.E. Beaumont ◽  
D.K. Berg ◽  
G.W. Asher
Keyword(s):  
Red Deer ◽  
Polar Body ◽  
Negative Control ◽  
Blastocyst Development ◽  
Cell Counts ◽  
First Polar Body ◽  
Significant Difference ◽  
Chi Square Analysis ◽  
Polar Body Extrusion

Successful activation of red deer oocytes is a necessary prerequisite for the cloning of red deer individuals with desirable genetic characteristics. To investigate this, an established biphasic protocol used for oocyte activation in sheep was investigated for suitability. The method chosen was 5μM Ionomycin for 5min followed by 2mM 6DMAP for 3h ( Loi P et al., 1998 Biol. Reprod. 58, 1177–1187). The medium used during activation and subsequent culture was Deer Synthetic Oviduct Fluid, which has been shown to support routine in vitro fertilization and blastocyst development (15%) of in vitro-matured red deer oocytes (DSOF, Berg D et al., 2003 Theriogenology 59, 189–205). Red deer abattoir-derived COCs were matured in vitro for 22h before random allocation across 3 treatment groups comprising a standard IVF group, the activation group and a negative control group exposed to medium only. Activation treatment oocytes were stripped of cumulus by vortexing in 0.1% hyaluronidase before selecting for first polar body extrusion. First-step activation was performed in medium comprising HEPES-buffered IVF-DSOF containing 4mM Ca2+. Second-step activation used 3mM Ca2+ early DSOF under 7% O2, 5% CO2, and 88% N2 at 38.5°C. Standard IVF was conducted at 23h post-IVM using 4mM Ca2+ IVF-DSOF and 0.5×106mL−1 final sperm concentration. Following activation and IVF, oocytes were washed 3 times in HEPES DSOF before culture for 7 days in sequential DSOF with late DSOF on Day 4 containing 1.5mM Ca2+. Cleavage was assessed 24h after activation, and all blastocysts were fixed for cell counts. Four replicates of each treatment were performed. Cleavage and blastocyst rates were examined by chi-square analysis and cell numbers by ANOVA. First polar body extrusion rate was 84%. Cleavage was similar between the activation treatment and IVF (P&gt;0.05 ); but a significant difference was found in blastocyst development rates (P&lt;0.05) with the Ionomycin and 6DMAP protocol being superior to the IVF treatment. Exposure to high Ca2+ media alone resulted in only 5% of the negative control oocytes cleaving to 2 cells. Results show that Ionomycin and 6DMAP are effective in activating red deer oocytes and DSOF is a suitable medium to produce parthenogenetic blastocysts.

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