39 BOVINE SOMATIC CELL NUCLEAR TRANSFER USING CUMULUS - OOCYTE COMPLEXES COLLECTED FROM THE IDENTICAL INDIVIDUAL BY OVUM PICKUP

2007 ◽  
Vol 19 (1) ◽  
pp. 138 ◽  
Author(s):  
K. Hasegawa ◽  
S. Takahashi ◽  
S. Akagi ◽  
K. Takeda ◽  
K. Imai ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Cumulus Cells ◽  
Single Strand Conformation Polymorphism ◽  
Blastocyst Stage ◽  
Polymorphism Analysis ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Developmental Rates ◽  
Vitro Development

We previously produced a cloned calf by nuclear transfer (NT) using cumulus cells removed from cumulus–oocyte complexes (COCs) after IVM. If both cumulus cells and oocytes are obtained identically and individually, and can be used simultaneously for NT, the production of cloned cows will be more expedient. And the cloned offspring produced from them will not exhibit the heteroplasmic mixed mtDNAs of donor cells and recipient oocytes. In this study, we examined the developmental potential of NT embryos using cumulus–oocyte complexes (COCs) collected from cows individually by ovum pickup (OPU). The cumulus cells were removed from COCs after IVM. The cumulus cells and cumulus-free MII oocytes derived from the same cow were used as donor nuclei and recipient oocytes, respectively. NT was performed as previously described (Akagi et al. 2003 Clon Stem Cells 5, 101–108). In Experiment 1, we examined the in vitro development of NT embryos using COCs collected by OPU. The aspiration of the follicles was performed once a week consecutively for 6 weeks in 6 cows (Cows A, B, C, D, E, and F) without hormone stimulation. In Experiment 2, we examined fetal development after the transfer of NT embryos. A Japanese black cow (Cow G) was used for OPU. On Day 7, 13 NT blastocysts were transferred to 7 recipient cows. The mtDNA genotypes of the donor cow and the cloned calf were analyzed by PCR-mediated single-strand conformation polymorphism analysis as previously described (Takeda et al. 2003 Mol. Reprod. Dev. 64, 429–437). The results of Experiment 1 are summarized in Table 1. Fusion rates did not differ among individual cows. However, the developmental rates of NT embryos at the blastocyst stage varied widely among individual cows, with a range of 19 to 64%. In Experiment 2, 2 of 7 recipient cows became pregnant on Day 30. One pregnant cow aborted on Day 60, and another cow calved a healthy calf. The mtDNA genotype of the cloned calf was confirmed to be identical with that of the donor cow. These results indicate that COCs from an identical individual can be used as donor nuclei and recipient oocytes for NT in order to produce female clones with the same mtDNA as that of the donor cow. Table 1.In vitro development of NT embryos using COCs collected by OPU

scholarly journals 43IMPROVED IN VITRO DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS WITH 6-DMAP FOLLOWING FUSION

2004 ◽  
Vol 16 (2) ◽  
pp. 144
Author(s):  
G.-S. IM ◽  
L. Lai ◽  
Z. Liu ◽  
Y. Hao ◽  
C.M. Murphy ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Developmental Rate ◽  
Cell Number ◽  
Blastocyst Stage ◽  
In Vitro Development ◽  
Electric Pulses ◽  
Developmental Rates ◽  
Vitro Development

Although nuclear transfer (NT) has successfully produced cloned piglets, the development to blastocyst and to term is still low. Activation of the NT embryos is one of the key factors to improve the developmental ability of porcine NT embryos. Electric pulses as well as chemicals have been used to activate porcine NT embryos. This study was conducted to investigate the effect of continued activation following fusion pulses on in vitro development of porcine NT Embryos. Oocytes derived from a local abattoir were matured for 42 to 44h and enucleated. Ear skin cells were obtained from a 4-day-old transgenic pig transduced with eGFP recombinant retrovirus. Enucleated oocytes were reconstructed and cultured in PZM-3 in a gas atmosphere of 5% CO2 in air. Cleavage and blastocyst developmental rates were assessed under a stereomicroscope on Day 3 or 6. Blastocysts were stained with 5μg of Hoechst 33342 and total cell number was determined with an epifluorescent microscope. In Experiment 1, oocytes were activated with two 1.2kV/cm for 30μs (E) in 0.3M mannitol supplemented with either 0.1 or 1.0mM Ca2+. In each treatment, activated oocytes were divided into three groups. The first group was control (E). Other two groups were exposed to either ionomycin and 6-DMAP (E+I+D) or 6-DMAP (E+D) immediately after the electric pulses. In Experiment 2, fusion was conducted by using 1.0mM Ca2+ in the fusion medium. Fused NT embryos were divided into three treatments. NT embryos were fused and activated simultaneously with electric pulse as a control (C); the second group was treated with 6-DMAP immediately after fusion treatment (D0); and the third group was treated with 6-DMAP at 20min (D20) after fusion. In experiment 1, for 0.1mM Ca2+, developmental rates to the blastocyst stage for E, E+I+D or E+D were 12.5, 26.7 and 22.5%, respectively. For 1.0mM Ca2+, developmental rates to the blastocyst stage were 11.4, 28.3 and 35.6%, respectively. The activated oocytes treated with 6-DMAP following the electric pulses by using 1.0mM Ca2+ in fusion medium had higher (P<0.05) developmental rates to the blastocyst stage. In Experiment 2, developmental rates to the blastocyst stage for C, D0 or D20 were 10.0, 12.3, and 19.9%, respectively. Developmental rate to the blastocyst stage was higher (P<0.05) in D20. Fragmentation rates were 19.9, 10.8, and 9.0%, respectively. Regardless of Ca2+ concentration in fusion medium, continued treatments with chemicals following electric pulses supported more development of porcine activated oocytes. Treating NT embryos with 6-DMAP alone after fusion was completed by using 1.0mM Ca2+ in fusion medium improved the developmental rates to the blastocyst stage and prevented fragmentation accompanied by electric fusion. This study was supported by NIH NCRR 13438 and Food for the 21st Century.

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

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

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

Effects of cycloheximide treatment and interval between fusion and activation on in vitro development of pig nuclear transfer embryos

2002 ◽  
Vol 14 (4) ◽  
pp. 191 ◽  
Author(s):  
M. A. Martinez-Diaz ◽  
K. Ikeda ◽  
Y. Takahashi
Keyword(s):  
Nuclear Transfer ◽  
Kinase Activity ◽  
Short Interval ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
In Vitro Development ◽  
M Phase ◽  
Cytostatic Factor ◽  
Vitro Development

The effects of cycloheximide (CHX) treatment and the interval between fusion and activation on the development of pig nuclear transfer (NT) embryos constructed with enucleated oocytes and serum-starved granulosa/cumulus cells were examined. One group of couplets was fused and activated simultaneously (FAS) by a single electrical pulse (activation pulse). Another three groups of couplets were fused electricaly 1.5, 2.5 or 4.5 h before being subjected to the activation pulse (FBA). Each group was divided into two subgroups and incubated with or without CHX. The NT embryos treated with CHX showed a high and stable cleavage rate, regardless of the interval between fusion and activation; however, development to blastocysts was improved only when the NT embryos were subjected to FAS with CHX. These results indicate that CHX-sensitive events occurring shortly after FAS may be responsible for the development to blastocysts. Fusion pulse rarely activated M II oocytes, but rapidly dropped the p34cdc2 kinase activity in NT embryos. A pronucleus-like structure was observed 2-2.5 h after the activation pulse with CHX in NT embryos of both the FAS and FBA groups. Therefore, successive inactivation of M-phase promoting factor and cytostatic factor at a certain short interval may also play an important role in the development of NT embryos.

35 EFFECTIVENESS OF MICROWELL-BASED IN VITRO CULTURE SYSTEMS FOR BOVINEZONA-FREE CLONED EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 124
Author(s):  
C. Feltrin ◽  
M. Machado ◽  
L. M. V. Queiroz ◽  
M. A. S. Peixer ◽  
P. F. Malard ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Aggregation State ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Vitro Development

In vitro embryo production by handmade cloning (HMC) usually requires individual embryo culture, because zona-free embryos cannot be grouped in standard in vitro culture (IVC) protocols. The aim of this study was to evaluate the developmental potential of bovine embryos produced by HMC (Ribeiro et al. 2009 Cloning Stem Cells 11, 377–386) after in vitro culture (IVC) in 3 microwell (WOW) systems. After in vitro maturation, oocytes were denuded and incubated in demecolcine (Ibáñez et al. 2003 Biol. Reprod. 68, 1249–1258), followed by zona pellucida removal, oocyte bisection, embryo reconstruction, electrofusion, and chemical activation. Cloned embryos were allocated to 1 of 3 IVC groups: cWOW: conventional microwells (250 μm, round; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264); mWOW: modified microwells (130 μm, conical; Feltrin et al. 2006 Reprod. Fert. Dev. 18, 126); and WOW-PDMS: microwells in polydimethylsiloxane chips (170 μm, cylindrical with microchannels); IVF embryos were used as controls (Bertolini et al. 2004 Reproduction 128, 341–354). Cleavage (Day 2), blastocyst (Day 7), and pregnancy (Day 30) rates were analysed by the chi-square test, for P < 0.05. Results are shown in Table 1. Cleavage rates were similar between groups, but development to the blastocyst stage was higher in IVF controls than cloned embryo groups. Among cloned embryo groups, blastocyst rate was higher in the mWOW group than the conventional and the PMDS-based microchannels. Nevertheless, in vivo development to Day 30 of pregnancy was not different between cloned groups. Our results for in vitro embryo development indicated that the mWOW provided more suitable conditions for embryo development to the blastocyst stage when compared with cWOW or even WOW-PDMS. Among some possible reasons include the physical advantage of a smaller microwell that may better mimic the constraining effect of the zona pellucida on the developing embryo. That may also provide greater blastomere stability, favouring the aggregation state during the first rounds of cleavages, also aiding compaction and subsequent cavitation. The narrower microwell system appeared to have promoted better in vitro development than the conventional and the DMPS-based microwell systems, with no impact on subsequent in vivo development. However, the IVC in the WOW-PDMS system supported reasonable rates of development, in accordance with the current literature. Table 1.In vitro development of bovine IVF and cloned embryos produced after the in vitro culture in distinct IVC systems

Development of porcine oocytes from preovulatory follicles of different sizes after maturation in media supplemented with follicular fluids

2000 ◽  
Vol 12 (4) ◽  
pp. 133 ◽  
Author(s):  
Ki-Won Yoon ◽  
Tae-Young Shin ◽  
Jong-Im Park ◽  
Sangho Roh ◽  
Jeong M. Lim ◽  
...  
Keyword(s):  
Blastocyst Stage ◽  
Developmental Potential ◽  
Large Follicle ◽  
In Vitro Development ◽  
Maturation Medium ◽  
Preovulatory Follicles ◽  
Vitro Development ◽  
Small Follicle ◽  
Follicular Fluids

The development of porcine oocytes from large (3.1–8.0 mm in diameter) or small (<3.1 mm) follicles was examined after maturation culture in medium containing porcine follicular fluid (pFF). Large follicles yielded larger (256 m v. 221 m; P<0.05) cumulus–oocyte complexes and more (22 v. 14%) morphologically normal oocytes than small follicles (Experiment 1). In Experiments 2–4, maturation media supplemented with mixed pFF (10%) from small and large follicles was used. More oocytes from large follicles matured (58% v. 91%), formed pronuclei (81% v. 90%) and developed to the blastocyst stage (2% v. 10%) than oocytes from small follicles. In Experiments 5–7, the effects of pFF collected from either small or large follicles on oocyte development were examined. Regardless of the source of oocytes, large-follicle-derived pFF more significantly enhanced preimplantation development than did small-follicle-derived pFF. The highest rate of blastocyst formation (16%) was found when oocytes from large follicles were cultured in maturation medium containing large-follicle-derived pFF. These results suggest that oocytes from large follicles have greater developmental potential than oocytes from small follicles, and that the origin of pFF, which is added to the maturation media, might be an important factor for improving in vitro development of porcine oocytes.

scholarly journals 25 IN VITRO DEVELOPMENT OF AGGREGATED NUCLEAR TRANSFERRED EMBRYOS DERIVED FROM BOVINE CUMULUS CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 162
Author(s):  
S. Akagi ◽  
B. Tsuneishi ◽  
S. Watanabe ◽  
S. Takahashi
Keyword(s):  
Zona Pellucida ◽  
Cumulus Cells ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
In Vitro Development ◽  
Functional Peptide ◽  
Vitro Development

It has been reported that aggregation of two nuclear transfer (NT) mouse embryos shows an improvement in full-term development (Boiani M et al. 2003 EMBO J. 22, 5304–5312). In this study, we examined the effect of aggregation on in vitro development of bovine NT embryos. As donor cells for NT, cumulus cells of passage 3–5 were used following culture in serum-starved medium for 5–7 days. NT was performed as previously described (Akagi S et al. 2003 Mol. Reprod. Dev. 66, 264–272). NT embryos were cultured in a serum-free medium (IVD-101, Research Institute of Functional Peptide Co., Ltd., Shimojo, Yamagat, Japan). Eight-cell-stage embryos on Day 2 or 16- to 32-cell-stage embryos on day 4 were used for embryo aggregation after removal of the zona pellucida. A small depression was made in a 25-μL drop of TCM-199 with 50 μg/mL phytohemagglutinin (TCM199/PHA) or IVD-101 using a darning needle. Two or three NT embryos were placed into the depression in the drop of TCM199/PHA for 20 min. NT aggregates were then moved into the depression in the drop of IVD-101 and cultured until Day 7. In vitro development of NT aggregates was summarized in Table 1. There were no differences in the cell number and ICM ratio of blastocysts between non-aggregated zona-intact and zona-free embryos. All aggregates of three NT embryos developed to the blastocyst stage and the cell number of these blastocysts was significantly higher than that of non-aggregated NT blastocysts. These results indicate that removal of the zona pellucida does not affect the cell number and ICM ratio of blastocysts and that aggregates of three NT embryos can develop to blastocysts with high cell numbers which are equivalent to in vivo-derived embryos (166 ± 11, Knijn HM et al. 2003 Biol. Reprod. 69, 1371–1378). Table 1. Development, cell number, and ICM ratio of NT aggregates

scholarly journals 55IN VITRO DEVELOPMENT OF ENUCLEATED DOMESTIC CAT OOCYTES RECONSTRUCTED WITH SKIN FIBROBLASTS OF DOMESTIC AND LEOPARD CATS

2004 ◽  
Vol 16 (2) ◽  
pp. 149 ◽  
Author(s):  
C. Lorthongpanich ◽  
C. Laowtammathron ◽  
S. Muenthaisong ◽  
T. Vetchayan ◽  
M. Ketudat-Cairns ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Cell Types ◽  
Blastocyst Stage ◽  
Skin Fibroblasts ◽  
Domestic Cat ◽  
Developmental Potential ◽  
A Cell ◽  
Vitro Development

The domestic cat is a valuable model for studies in assisted reproductive technology in felid species. Therefore, in this experiment we evaluated the in vitro developmental potential of enucleated domestic cat oocytes reconstructed with somatic cells from domestic and leopard cats. Skin fibroblasts were isolated from female domestic and leopard cats. The oocytes were collected by aspiration of follicles from ovaries that were superovulated with 200IU PMSG. In vitro-matured oocytes were enucleated and individual donor cells (diameter 14–16μm) were inserted into the perivitelline space of the enucleated oocyte. Fusion was performed at 26–27h post-maturation by placing a cell-oocyte couplet between both tips of the needle electrode and electrostimulating with a 2-DC pulse (30V, 30μs) in fusion medium containing 0.3M Mannitol+0.1mM MgCl2. Activation was performed 1 to 2h post-fusion by incubation in 7% ethanol at room temperature for 5min followed by cultured in 10μgmL−1 cycloheximide and 1.25μgmL−1 cytochalasin D at 38°C in 5% O2, 5% CO2, 90% N2 conditions. After activation, the reconstructed embryos were cultured in 100-μL droplets of Tyrode’s medium (Gomez et al., 2003 Theriogenology 60, 239–251.) supplemented with 0.3% BSA at 38°C in a 5% O2, 5% CO2, 90% N2 environment for 2d. Then, 8-cell embryos were cultured in 100-μL droplets of Tyrode’s medium supplemented with 10% FCS at 38°C in a 5% O2, 5% CO2, 90% N2environment for 5d. The cleavage rates of oocytes reconstructed with either donor cell types were not different. The percentages of blastocyst formation from parthenogenotes and nuclear transfer embryos derived from domestic cat fibroblasts (8/56, 14.3% and 7/51, 13.7%, respectively) were significantly higher than that for nuclear transfer embryos constructed with leopard cat fibroblasts (3/45, 6.7%). These results indicate that enucleated domestic cat oocytes reconstructed with skin fibroblasts of leopard cats can develop to the blastocyst stage. This experiment was supported by Suranaree University of Technology. Table 1 In vitro development of domestic cat oocytes reconstructed with domestic and leopard skin fibroblasts and parthenogenetic activation

scholarly journals 99 COMPARISON OF IN VITRO DEVELOPMENT FOLLOWING CRYOPRESERVATION OF MEISHAN AND WHITE CROSS SWINE EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 200
Author(s):  
B. White ◽  
M. Montagner ◽  
G. Mills ◽  
P. Gonçalves ◽  
R. Christenson
Keyword(s):  
Blastocyst Stage ◽  
Swine Industry ◽  
In Vitro Development ◽  
Embryo Culture Medium ◽  
Frozen Embryos ◽  
Developmental Rates ◽  
Fine Glass ◽  
Vitro Development ◽  
And Control

Development of improved protocols for cryopreservation of zona pellucida-intact porcine embryos could greatly impact the swine industry. Our aim was to investigate in vitro development following cryopreservation of embryos from Chinese Meishan (M) and occidental white cross (WC) breeds using a modified protocol described previously (Misumi K et al. 2003 Theriogenology 60, 253–260). First-parity M sows (n = 11) and WC gilts (n = 13) were observed for estrus every 12 h and inseminated at 12 and 24 h after estrous onset within breed using semen from 2 different boars. Females were sacrificed between Days 4.5 and 6 after estrus and embryos were collected using Beltsville embryo culture medium (BECM). Compact morula (CM) or blastocyst stage embryos from each female within breed were randomly allocated either directly into the culture system to serve as controls (68 M and 48 WC embryos) or to undergo cryopreservation. A total of 101 M and 78 WC embryos were cryopreserved using the following protocol: (1) 5 min in BECM + 10% ethylene glycol (EG); (2) 5 min in BECM + 10% EG + 0.27 M sucrose + 1% polyethylene glycol (PEG); and (3) 30 to 45 s in BECM + 40% EG + 0.36 M sucrose + 2% PEG. In the last solution, 5 to 10 embryos in a 5- to 10-μL microdrop attached to a fine glass pipette were exposed to the vapor phase of liquid nitrogen (LN2) for 15 s and then plunged into LN2. The pipette tip was broken and the tip and associated frozen microdrop were placed inside an LN2-submerged 2-mL cryotube containing a hole in the lid for 1 h. Next, embryos were thawed using a 4-step (5 min each) procedure: (1) BECM + 5% EG + 0.57 M sucrose; (2) BECM + 2.5% EG + 0.29 M sucrose; (3) BECM + 0.3 M sucrose; and (4) BECM alone. All procedures were performed with solutions maintained at 37°C. Cryopreserved and control embryos were cultured in 50 μL drops of modified Whitten's medium + 1.5% BSA under oil at 37°C in a 5% CO2 in air environment and scored daily for development. For embryos undergoing cryopreservation, retrieval rates from cryovials were 92% and 96% for M and WC, respectively. The percentage of embryos surviving 24 h after cryopreservation without lysis or degeneration was higher for M (72%) than for WC (44%; P < 0.001; χ2-test). However, in vitro development of embryos that survived cryopreservation was not different between M and WC at the expanded (64%) or hatched (22%) blastocyst stages. Developmental rates were significantly higher for control embryos than for frozen embryos from both breeds. Rates of expanded blastocyst formation did not differ between M and WC control embryos (98% and 95%, respectively), but more M embryos developed to the hatched blastocyststage (22% for M v. 9% for WC; P < 0.05). Our results suggest that M embryos have a higher capacity to survive the vitrification process than WC embryos. Funding for M. Montagner was provided by CAPES, Brazil.

scholarly journals 142 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS CULTURED IN KSOM, CR1AA, OR KSOM/CR1aa

2005 ◽  
Vol 17 (2) ◽  
pp. 221 ◽  
Author(s):  
M.R.B. Mello ◽  
C.E. Ferguson ◽  
A.S. Lima ◽  
M.B. Wheeler
Keyword(s):  
Embryo Culture ◽  
Cumulus Cells ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Cell Stage ◽  
In Vitro Development ◽  
Significant Difference ◽  
Developmental Rates ◽  
Vitro Development

In vitro embryo culture is an important step of in vitro production of bovine embryos. It has been shown that IVF-derived bovine embryos cultured in KSOM or CR1aa have high development rates. In our laboratory, we have observed that 8-cell embryos are morphologically superior when embryos are cultured in KSOM whereas blastocysts are morphologically superior when embryos are cultured in CR1aa. Based on these observations, we hypothesized that development of IVF-derived bovine embryos can be improved by sequential use of these media (KSOM and CR1aa). The aim of this experiment was to compare the in vitro development of bovine embryos cultured in KSOM, CR1aa or KSOM/CR1aa supplemented with BSA at Day 0 and BSA and FBS at Day 3. In order to accomplish the sequential culture, fertilized oocytes where cultured in KSOM to the 8-cell stage and then transferred to CR1aa for further development. Oocytes were purchased from Bomed (Madison, WI, USA), and after 22 hours of maturation were fertilized with frozen-thawed semen for 5 hours at 39°C in 5% CO2. After fertilization, the presumptive zygotes were denuded from cumulus cells by votexing and were randomly allotted to one of 3 treatments: (1) cultured only in KSOM (n = 110), (2) cultured only in CR1aa (n = 102), and (3) cultured in KSOM in the first 3 days and then in CR1aa from Day 3 to Day 9 (n = 110). The embryo culture was carried out in 50-μL droplets of medium that were placed in an airtight modular incubator filled with 5% CO2, 5% O2 and 90% N2. The embryos were evaluated on Days 6 to 9 post insemination. All embryo developmental rates were calculated from presumptive zygotes. The Day 6 morula rates were 52%, 40%, and 47% for KSOM, CR1aa, and KSOM/CR1aa, respectively. The Day 7 blastocyst rates for KSOM (40%), CR1aa (25%), and KSOM/CR1aa (30%) were not significantly different; however, Day 9 hatched blastocyst rates were significantly higher (P < 0.05) for KSOM (22%) compared to CR1aa (9%) but not different from KSOM/CR1aa (14%). Regarding embryo quality, Day 7 transferable embryos rates (Grade 1 and Grade 2) were 35%, 25%, and 30%, respectively for KSOM, CR1aa, and KSOM/CR1aa; however, no significant difference was observed. These results indicate that IVF-derived bovine embryos can develop in KSOM, CR1aa, or KSOM/CR1aa with no significant difference among morula, blastocyst and hatched blastocyst rates. However, the combination of KSOM and CR1aa during in vitro culture did not decrease the morula and blastocyst rates.

78 EFFECTS OF DIFFERENT ACTIVATION PROTOCOLS ON ACTIN FILAMENT DISTRIBUTION AND IN VITRO DEVELOPMENT OF MINIATURE PIG NT EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 147
Author(s):  
K. Yamanaka ◽  
S. Sugimura ◽  
T. Wakai ◽  
T. Shoji ◽  
H. Sasada ◽  
...  
Keyword(s):  
Actin Filament ◽  
Nuclear Transfer ◽  
Subsequent Development ◽  
Miniature Pig ◽  
Physical Damage ◽  
In Vitro Development ◽  
Fetal Fibroblasts ◽  
Developmental Rates ◽  
Vitro Development

In the process of producing reconstructed oocytes nuclear transfer (NT) embryos by somatic cell nuclear transfer, in vitro-matured oocytes can be used as recipient ones. It, however, has been well documented that after IVF porcine embryos derived from in vitro-matured oocytes have a small number of cells and low viability compared from those in vivo. As one possible reason, abnormal actin filament distribution has been detected in abnormal embryo cleavage and small cell numbers (Wang et al. 1999 Biol. Reprod. 60, 1020-1028). Artificial activation, which is necessary for development of NT embryos, can affect actin filament distribution of porcine oocytes matured in vitro, resulting in fragmentation (Kawahara et al. 2002 Theriogenology 58, 1081-1095). In the present study, we investigated effects of different activation protocols on actin filament distribution and in vitro development of miniature pig NT embryos. Porcine oocytes collected from ovaries were matured in vitro for 40 to 44 h in NCSU-23. First, we compared different activation protocols in development rates to blastocysts of oocytes activated. We used three activation methods (15 �M ionomycin treatment for 20 min (I), double DC pulses of 1.2 kV/cm for 60 ms in intervals of 5 s (E), and 5 mg/mL cycloheximide treatment for 5 h (C)) to prepare seven activation protocols (I, E, C, I + C, I + E, E + C, and I + E + C). Second, we examined effects of different activation protocols on actin filament distribution and subsequent development of NT embryos activated by the different activation protocols. Matured oocytes were enucleated, and fused with miniature pig fetal fibroblasts in calcium-free medium; approximately 3 h later, the resultant NT embryos were activated with three activation protocols (E, I + C, or I + E + C). All data were analyzed by chi-square test. The developmental rates to blastocysts in the I, E, C, I + C, I + E, E + C, and I + E + C groups were 5.6, 11.1, 0.0, 36.1, 20.7, 14.6, and 24.7%, respectively, showing that the rate in oocytes activated with I + C was significantly higher (P < 0.05) than the rates in oocytes activated by other treatments. In NT embryos, the developmental rates to blastocysts in the E, I + C, or I + E + C groups were 4.1, 14.3, and 4.6%, respectively, showing that the rate in NT embryos activated with I + C was significantly higher (P < 0.05) than the rate in NT embryos activated with other treatments. The abnormal rate of actin filament distribution in NT embryos activated with E or I + E + C was significantly higher (P < 0.05) than that in NT embryos activated with I + C (26.7% or 33.3% vs. 6.7%). The present results suggest that in miniature pig NT embryos an activation protocol by ionomycin combined with cycloheximide treatments may avoid physical damage to actin filaments with the resultant improvement of subsequent development.

Sign in / Sign up

Export Citation Format

Share Document