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).

32 PREGNANCY OF EQUINE CLONED EMBRYOS MICROINJECTED WITH PLURIPOTENCY INDUCING GENES (Oct4, Sox2, c-Myc, K1f4)

2014 ◽  
Vol 26 (1) ◽  
pp. 130
Author(s):  
R. Olivera ◽  
R. Jordan ◽  
C. Alvarez ◽  
M. Radrizzani ◽  
G. Vichera
Keyword(s):  
Pregnancy Rate ◽  
Electric Pulse ◽  
Polar Body ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Pregnancy Rates ◽  
Control Group ◽  
Blastocyst Development ◽  
Donor Nucleus ◽  
First Polar Body

Animal cloning is a high impact tool for scientific and economical production, but still with inefficient results. The efficiency of the cloning process depends on the state of differentiation of the donor cell. An adult equine somatic cell can be differentiated to a pluripotent stem cell (iPSC) inducing the expression of certain transcription factors (Oct4, Sox2, c-Myc, and K1f4; Breton et al. 2013). The objective of this work was to assess the effect of the intracytoplasmic injection of pluripotency inducing genes on embryo development and pregnancy rates of equine cloned embryos. Cumulus–oocyte complexes (COC) were obtained from slaughterhouse ovaries. Oocyte collection and maturation procedure were performed as described by Lagutina et al. (2007). After the removal of cumulus cells, oocytes showing first polar body were microinjected with a mixture 1/3 of plasmids/liposomes (Mi group). The plasmid used was the pEP4-E02s-EM2k, which encodes the human genes Oct4, Sox2, Myc, and K1f4. The DNA concentration was adjusted to 0.5 μg mL–1. Microinjected oocytes were enucleated using the zona free method. Adult male skin fibroblasts from the same animal were used as donor nucleus cells. These fibroblasts were attached to the ooplasts with phytohemagglutinin and then fused with an electric pulse. Activation was performed using 8.7 mM ionomycin for 4 min, followed by culture for 4 h in a combination of 1 mM 6-DMAP and 5 mg mL–1 cycloheximide. Zona free reconstructed embryos (ZFRE) were cultured for 7 to 8 days in DMEM-F12 in the well of the well (WOW) system, aggregating 3 embryos per well. A control group (CC group) of not microinjected embryos was included. Cleavage and blastocyst development was assessed at Days 2 and 7, respectively. Transcervical transfer of 49 Day 7 to 8 blastocysts was performed 6 days after ovulation. The mares received 2 blastocysts per transfer. Pregnancy was diagnosed by transrectal ultrasonography 15 days after ovulation. Cleavage and blastocyst rates were analysed by Chi-squared test and pregnancy rate by Fisher test (P < 0.05). Cleavage was 92.1% (n = 58/63) for the Mi group and 90.4% (n = 868/960) for the CC group. Blastocyst rate was statistically higher per well, 28.6% (n = 6/21) v. 13.4% (n = 43/320) but not per oocyte, 9.5% (n = 6/63) v. 4.5% (n = 43/960), for the Mi and CC groups, respectively. Pregnancy rate was 17% (n = 1/6) for the Mi group and 7% (n = 3/43) for the CC group. No twin pregnancies were found and all the pregnancies are still ongoing. The higher blastocyst rates obtained with the embryos microinjected with pluripotency inducing genes compared with the control group showed an improvement in embryo quality. In conclusion, the data presented indicate that the intracytoplasmic microinjection of pluripotency inducing genes in equine zona free cloned embryos improved blastocyst rates on a per well basis and showed a tendency to improve the pregnancy rates. The expression of the Oct4, Sox2, c-Myc, and K1f4 genes could be probably generating better reprogrammed donor nucleus compared with adult differentiated cells used in conventional cloning.

201 ENDOSCOPE-GUIDED TRANSFER OF SPERM-INJECTED OOCYTES INTO THE OVIDUCTS OF ELAND AND BONGO ANTELOPES

2010 ◽  
Vol 22 (1) ◽  
pp. 259 ◽  
Author(s):  
G. Wirtu ◽  
R. MacLean ◽  
J. Galiguis ◽  
D. Paccamonti ◽  
B. Eilts ◽  
...  
Keyword(s):  
Assisted Reproductive Technologies ◽  
Transvaginal Ultrasound ◽  
Polar Body ◽  
Prostaglandin F2α ◽  
Reproductive Technologies ◽  
Pelvic Cavity ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
First Polar Body ◽  
The Right

We have previously established methods of gamete collection in Eland and Bongo antelopes; however, blastocyst development following IVF, intracytoplasmic sperm injection (ICSI), or somatic cell nuclear transfer (SCNT) has been sporadic, thus few developmentally competent embryos have been produced for uterine transfer. In the present study, we evaluated the possibility of oviductal transfer of presumptive zygotes. Estrous cycle synchronization and ovarian superstimulation were done as follows. Females were treated with altrenogest (0.11 g/os daily) for 7 days. The FSH Folltropin® was dissolved in 30% polyvinylpyrrolidone and administered, i.m. on Days 5 and 7 of altrenogest treatment at 266 mg and 134 mg, respectively. Prostaglandin F2α (Lutalyse®, 25 mg, i.m.) was administered on Day 7. Transvaginal ultrasound-guided oocyte retrieval was conducted on Day 9 as described previously (Wirtu G et al. 2009 Anim. Reprod Sci. 111, 160-172). Eleven oocytes were recovered from 2 Elands and 4 from 1 Bongo. One oocyte of each species was degenerate at the time of recovery. Oocytes were subjected to IVM for approximately 22 h, when Piezo drill-assisted ICSI was done using frozen-thawed Eland or Bongo spermatozoa. Oocytes were activated (5% ethanol, 5 min) and cultured overnight in CR1aa medium supplemented with BSA. Presumptive zygotes were subjected to endoscopic oviductal transfer at 21 to 24 h after ICSI. The oviductal transfer was adapted from the technique developed in domestic cattle (Wetscher F et al. 2005 Theriogenology 64, 30-40). Animals were sedated by i.m. administration of xylazine HCl and butorphanol tartrate and restrained in a hydraulic chute (Tamer®) that was used to squeeze and lift the females. An epidural block (5 mL of lidocaine) was induced after cleaning the rectum, perineal region, and the injection site. A uterine relaxant, isoxsuprine HCl (10 mg, i.v), was then administered. The Brem/Besenfelder set for laparoscopic bovine embryo transfer (Karl Storz Endoscope, Karl Storz GmbH & Co. KG, Tuttlingen, Germany) was used for the oviductal transfer. Briefly, a cannula (12.5 mm in diameter, 49.5 cm long) fitted with a blunt-tip obturator was introduced into the vagina and positioned, during transrectal manipulation, at the mid-dorsal aspect of the vaginal vault. The blunt-tip obturator was replaced with a sharp-tip obturator, which was used to puncture through the vaginal wall for entry into the pelvic cavity. The trocar was subsequently replaced with an inner sheath containing a telescope (5.5 mm in diameter, 54 cm long) and a pre-loaded transfer tubing. Visualization of the ovaries and oviducts required insufflation of the peritoneal cavity. Transfer was done into the oviduct ipsilateral to the recently ovulating ovary. Ten Eland oocytes had extruded the first polar body and 8 that survived ICSI were transferred into the right oviduct of an Eland female. Two Bongo oocytes had extruded the first polar body; both survived ICSI and were transferred to the right oviduct of a Bongo female. Pregnancy diagnosis is pending. This minimally invasive method of accessing the oviduct has the potential to advance the application of assisted reproductive technologies in large nondomestic ungulates.

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.

32 CRYOPRESERVATION OF DONOR CELLS FOR NUCLEAR TRANSFER: EFFECT OF CELL FREEZING METHOD ON THE EFFICIENCY OF SOMATIC CELL NUCLEAR TRANSFER IN PIGS

2006 ◽  
Vol 18 (2) ◽  
pp. 125
Author(s):  
J. Estrada ◽  
E. Lee ◽  
J. Piedrahita
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Fusion Rate ◽  
Blastocyst Development ◽  
Freezing Method ◽  
Donor Cells ◽  
First Polar Body ◽  
Cell Freezing

Donor cell quality is one of the most important factors affecting somatic cell nuclear transfer (SCNT) in mammals. Many studies have been carried out to improve the donor cell characteristics in nuclear transfer, including studies on cell type, cell cycle stage, cell passage, and handling of donor cells before the SCNT. Even though most SCNT work is done with donor cells that have been previously frozen and thawed, no studies have been conducted to evaluate the effect of the cell freezing rate on the SCNT efficiency. The objective of this experiment was to evaluate the effect of the cell freezing method on development of pig SCNT embryos in vitro. Fibroblasts were collected from a 29-day-old female fetus, suspended in DMEM-F12 + 40% fetal bovine serum (FBS) + 10% dimethyl sulfoxide (DMSO), and placed in 1.6-mL cryovials for freezing. Vials were randomly assigned to two treatments: In treatment 1, cells were frozen at a controlled rate of 1�C/min in a programmable machine (P) until -40�C, and then plunged into liquid nitrogen (LN2; -196�C). In treatment 2, the traditional system (T), vials were placed in a styrofoam box and left overnight in a freezer at -80�C. The next day samples were plunged into LN2 (196�C). For each treatment, cells were thawed and cultured until confluence before being used for SCNT. Cells were used at passages 2 and 6. Cumulus-oocyte complexes (COCs) were aspirated from slaughterhouse ovaries and cultured for 39 h in TCM 199 supplemented with 10% porcine follicular fluid (pFF), 5 �g/mL insulin, 10 ng/mL epidermal growth factor (EGF), 0.6 mM cysteine, 0.2 mM pyruvate, 25 �g/mL gentamycin and 5 �g/mL each of equine and human chorionic gonadotropin (eCG and hCG). Oocytes were stained with bisbenzimide and enucleated in manipulation media with 7.5 �g/mL cytochalasin B by removing the first polar body and metaphase plate by means of a 16-�m beveled glass pipette. Cells from each treatment were injected into the perivitelline space of recipient enucleated oocytes and fused by two DC pulses of 140 V for 50 �s in fusion media. The fusion rate was evaluated 1 h later, and reconstructed oocytes were activated by two DC pulses of 120 V for 60 �s. After activation, oocytes were placed in bicarbonate-buffered NCSU-13 with 0.4% BSA and cultured at 38.5�C, 5% CO2 in a humidified atmosphere. Embryos were observed for cell cleavage at Day 2, and blastocyst development rate and cell number counting were done at Day 7 of culture. Every experiment was repeated three times. The temperature descending rate for P was slower and more linear (1�C/min vs. 2�C/min) than for the T method. Fusion rate was not significantly affected (P < 0.05) by the freezing method when they were evaluated either individually at each passage or accumulated regardless the passage (78.9 � 3.6% vs. 79.4 � 6.3%) for P and T, respectively. The same trends were observed for cleavage (61.2 � 5.2% vs. 64.3 � 5.2%), blastocyst development (4.2 � 1.8% vs. 5.0 � 2.8%), and number of cells at the blastocyst stage (19.4 � 3.1 vs. 19.8 � 6.2) for P and T, respectively. The present findings indicate that blastocyst development after SCNT does not differ when fetal fibroblasts donor cells are frozen by the two methods tested.

Fate of the germ cells in mammalian ovary: A review

2020 ◽  
Vol 3 ◽  
pp. 3
Author(s):  
Pramod K. Yadav ◽  
Anumegha Gupta ◽  
Alka Sharma ◽  
Anil Kumar Yadav ◽  
Meenakshi Tiwari ◽  
...  
Keyword(s):  
Germ Cells ◽  
Assisted Reproductive Technologies ◽  
Mammalian Species ◽  
Polar Body ◽  
Reproductive Technologies ◽  
Fetal Life ◽  
Oocyte Activation ◽  
Follicular Atresia ◽  
First Polar Body ◽  
Reproductive Life

Ovary has a fix number of germ cells during fetal life in mammals. The germ cells are depleted rapidly and a large number of germ cells (≥99%) are eliminated from the cohort of ovary through follicular atresia during prepubertal life. The various cell death pathways including apoptosis, autophagy, necrosis, and necroptosis are involved in follicular atresia. Hence, <1% of germ cells are culminated into oocytes that are available for meiotic maturation and ovulation during entire reproductive life. These oocytes are arrested at diplotene stage of meiotic prophase-I and remain arrested for few months to several years during entire reproductive life. Resumption from diplotene arrest in follicular oocytes starts in response to gonadotropins surge and progresses through metaphase-I to metaphase-II stage that extrudes first polar body at the time of ovulation. Surprisingly, oocytes do not wait for fertilizing spermatozoa and quickly undergo abortive spontaneous oocyte activation (SOA) in few mammalian species including humans. The abortive SOA makes oocyte unfit for fertilization and limits assisted reproductive technologies outcome. Indeed, majority of germ cells and oocytes are eliminated from the cohort of ovary and only few oocyte that are of good quality get selectively recruited to become right gamete after ovulation during entire reproductive life span in mammals.

scholarly journals Vitrification of immature oocytes of goats in Bangladesh

2019 ◽  
Vol 35 (1-2) ◽  
pp. 7-12
Author(s):  
MN Sharif ◽  
SM Choudhury ◽  
MM Rahman ◽  
MM Rahman ◽  
NS Juyena ◽  
...  
Keyword(s):  
Culture Medium ◽  
Assisted Reproductive Technologies ◽  
Polar Body ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Step Procedure ◽  
Inverted Microscope ◽  
Maturation Rate ◽  
Humidified Air

Cryopreservation of oocytes and embryos by vitrification can have advantages in assisted reproductive technologies (ARTs) in mammals. The aim of this study was to establish an effective vitrification procedure and cryodevice for goat’s oocytes in Bangladesh. Cumulus oocyte complexes (COCs) were collected from ovaries from slaughterhouse. COCs with more than 3 layers of cumulus cells were selected. COCs were vitrified by two-step procedure using 7.5% and 15% dimethyl sulphoxide (DMSO) as cryoprotective agent (CPA), loaded on Cryotop or French mini-straw, then directly plunged into liquid nitrogen (LN2). Then the COCs containing Cryotop or French mini-straws were warmed in 0.25 M sucrose and 20% FBS-supplemented tissue culture medium (TCM) 199 followed by in vitro culture in 50 μl droplets of bicarbonate-buffered TCM 199 supplemented with 10% FBS, pyruvate, FSH and oestradiol for 24 h at 39°C with 5% CO2 in humidified air. After maturation culture, oocytes were denuded and examined under inverted microscope for presence of polar body as the indication of maturation. The in vitro maturation rate of goat’s oocytes after vitrification and warming was 39.3 ± 6.8%, 31.3 ± 9.4%, 61.6 ± 14.2% when using Cryotop (cryodevice), French mini-straws and without vitrification (control), respectively. Maturation rate was significantly higher (P<0.05) without vitrification. It is suggested that both Cryotop and French mini-straw are efficient cryodevices for vitrification of goat’s oocytes and further investigation is required to optimize the protocol for vitrification and warming procedure for the satisfactory survival of goat’s oocytes. The Bangladesh Veterinarian (2018) 35(1&2): 7-12

372 SPERM ASTER FORMATION AND BLASTOCYST DEVELOPMENT OF IN VIVO-MATURED BOVINE OOCYTES FERTILIZED BY INTRACYTOPLASMIC SPERM INJECTION

2007 ◽  
Vol 19 (1) ◽  
pp. 301 ◽  
Author(s):  
T. Horiuchi ◽  
M. Takenaka ◽  
C. Kani ◽  
C. Emuta ◽  
Y. Ogata ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Blastocyst Development ◽  
Chi Square ◽  
First Polar Body ◽  
Bovine Oocytes ◽  
Sperm Aster

In cattle, activation treatment after intracytoplasmic sperm injection (ICSI) is required to improve cleavage and blastocyst rates (Horiuchi et al. 2002 Theriogenology 57, 1013–1024). The reason why the exogenous activation treatment in bovine ICSI is needed to promote cleavage and blastocyst development is not clear. The objective of this study was to examine the effect of activation treatment on sperm aster formation, cleavage, and blastocyst development of in vivo- and in vitro-matured bovine oocytes following ICSI. In vivo-matured oocytes were collected using transvaginal devices under ultrasound guide at about 29 h after GnRH injection from Japanese Black cows superstimulated with a total 19 mg FSH (Antrin�; Denka Pharmaceutical Co., Kanagawa, Japan) divided into twice daily over 3 days, and treated with 750 �g cloprostenol (Estramate�; Sumitomo Chemical Co., Tokyo, Japan). In a total of 8 aspiration sessions, 131 oocytes were collected; of 116 oocytes with expanded cumulus cells, 84 (72%) had a first polar body and were used for ICSI. On the other hand, in vitro-matured bovine oocytes were prepared by culturing immature follicular oocytes derived from abattoir ovaries. Bull spermatozoa, immobilized by scoring their tails, were injected into in vivo- or in vitro-matured oocytes. At 4 h after ICSI, the oocytes were treated with or without 7% ethanol for 5 min for activation. The injected oocytes were fixed at 8 h after ICSI, and sperm aster formation was examined by using specific antibodies and immunofluorescence microscopy. Data were analyzed by the chi-square test in all experiments. The rate of sperm aster formation in in vivo-matured oocytes was similar regardless of activation treatment (71% vs. 65%), but the rate in in vitro-matured oocytes was significantly (P &lt; 0.05) higher in the group receiving activation treatment than in the non-activation group (57% vs. 19%). Cleavage (88% vs. 88%) and blastocyst rates (59% vs. 47%) of in vivo-matured oocytes after ICSI were also similar, regardless of activation treatment, but cleavage (72% and 20%) and blastocyst rates (19% and 7%) of in vitro-matured oocytes were significantly (P &lt; 0.05) higher in the group receiving activation treatment than in the non-activation group. Moreover, the blastocyst rate of in vivo-matured oocytes was significantly (P &lt; 0.05) higher than the rate in in vitro-matured oocytes. These results show that activation treatment after ICSI of in vivo-matured bovine oocytes is not necessary for cleavage and blastocyst development, and suggest that the necessity of activation treatment in bovine ICSI has relevance to in vitro maturation of bovine oocytes.

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.

58 DEVELOPMENT OF α1,3-GALACTOSYLTRANSFERASE KNOCK-OUT CLONED PIG EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 187
Author(s):  
S. S. Hwang ◽  
M. R. Park ◽  
J. H. Shim ◽  
B. C. Yang ◽  
Y. G. Ko ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Fibroblast Cell ◽  
Cell Number ◽  
Normal Donor ◽  
Blastocyst Development ◽  
Step Method ◽  
Knock Out ◽  
Electric Pulses

This study was performed to increase the developmental rate of cloned embryos with the 1,3-Galactosyltransferase (GalT) gene knocked out (KO). Ovaries were collected from local slaughterhouse and immature oocytes were cultured in TCM-199 + 0.1% PVA + FSH + LH (0.5 μg mL-1) + EGF (10 ng mL-1) + 10% porcine follicular fluid (pFF) at 38.5°C in 5% CO2 humidified chamber for 40 h (1-step) or 20 h (with hormone) +20 h (without hormone; 2-step). After IVM, the oocytes with 1st polar body were enucleated and transferred the GalT KO donor cell originated from miniature pig. The embryos transferred with normal mini-pig ear fibroblast cell were used as control. The reconstructed embryos were fused with 2 electric pulses (DC) of 1.2 kV cm-1 for 30 μs. For the development of cloned embryos, the embryos were cultured in PZM-3 under 5% CO2 in air at 38.5°C for 6 days. The embryos were transferred to a surrogate (Landrace) at an earlier stage of the estrus cycle, and pregnancy diagnosis was determined at 28 days after embryo transfer using ultrasonography. Differences among treatment means were determined by a chi-square test. A probability of P < 0.05 was considered statistically significant. The maturation rate was significantly higher in the 2-step method (89.8 ± 2.75) compared with single maturation method (79.6 ± 8.95; P < 0.05). The blastocyst development of cloned embryos reconstructed with GalT KO donor cell (28.4 ± 2.14) was not different from cloned embryos by normal donor cell (27.4 ± 0.01). The cell number of GalT blastocyst (36.1 ± 11.1) was not different statistically from control (26.9 ± 9.3). The apoptosis rate was also not different in both groups (2.9 to 4.9%). Five surrogates were pregnant and the GalT KO fetuses were still ongoing pregnancy at 45 days after embryo transfer. This work received grant support from the Agenda Program (No. 200901FHT010305146 and No. 200901FHT010305535), Rural Development Administration, Republic of Korea.

32 EFFECTS OF FUSION/ACTIVATION METHODS ON DEVELOPMENT OF EMBRYOS PRODUCED BY NUCLEAR TRANSFER OF PORCINE FETAL FIBROBLASTS

2007 ◽  
Vol 19 (1) ◽  
pp. 134
Author(s):  
P. Q. Cong ◽  
E. S. Song ◽  
E. S. Kim ◽  
Z. H. Li ◽  
Y. J. Yi ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Metaphase Plate ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Pulse Number ◽  
Donor Cells ◽  
First Polar Body ◽  
Fetal Fibroblasts

Pigs have become increasingly important in the field of biomedical research, and interest has grown in the use of transgenic cloned pigs as potential xenograft donors. The present study were carried out to investigate the effects of intensity of DC pulse, number of DC pulses, and equilibration before fusion/activation on developmental ability of porcine embryos derived from nuclear transfer. Porcine cumulus-oocyte complexes (COCs) were cultured in modified TCM-199 (mTCM-199) medium for 44 h at 38.5�C, 5% CO2 in air. After in vitro maturation (IVM), metaphase II oocytes were selected for enucleation. Porcine fetal fibroblasts were obtained from a porcine fetus on Day 35 of gestation as donor cells. Oocytes were enucleated by removing, with a micropipette, the first polar body along with adjacent cytoplasm containing the metaphase plate; then a donor cell was injected in contact with the cytoplasm of each oocyte. In experiment 1, several different fusion/activation intensities (two DC pulses of 0.4, 0.8, 1.2, 1.6, and 2.0 kV cm-1 for 30 �s) were carried out to investigate the effect on the development of nuclear transfer embryos. In experiment 2, the reconstructed oocytes were fused and activated with 1, 2, or 3 DC pulses of 1.2 kV cm-1 for 30 �s. In experiment 3, reconstructed oocytes were equilibrated in mTCM-199 medium at 38.5�C, 5% CO2 for 0, 1, 2, 3, 4, 5, and 6 h. After equilibration, the reconstructed oocytes were fused and activated with one DC pulse of 1.2 kV cm-1 for 30 �s in fusion medium. The reconstructed embryos were transferred into PZM-3 medium containing 0.3% BSA for further culture. The rates of embryo cleavage and development of blastocyst stage were evaluated at 48 h and 6-7 days, respectively. The cell numbers of blastocysts were counted by using Hoechst 33342 epifluorescence staining. Data were analyzed by ANOVA and Duncan

Sign in / Sign up

Export Citation Format

Share Document