scholarly journals 32 EFFICIENCY OF FEMALE-DERIVED DONOR CELLS ON HIGH POSTNATAL SURVIVAL IN PIG CLONING

2005 ◽  
Vol 17 (2) ◽  
pp. 166
Author(s):  
S.K. Cho ◽  
M.R. Park ◽  
D.N. Kwon ◽  
E.K. Lee ◽  
S.J. Kang ◽  
...  
Keyword(s):  
Adenosine Monophosphate ◽  
Cell Number ◽  
Developmental Competence ◽  
Cell Stage ◽  
Male And Female ◽  
Electric Pulses ◽  
Donor Cells ◽  
Fetal Fibroblasts

The present study was conducted to investigate the developmental competence of male and female somatic cell derived nuclear transfer (NT) porcine embryos and also the production and survival efficiency of cloned male and female piglets. Maturation of porcine COCs was accomplished by incubation in NCSU-23 medium supplemented with 0.6 mM cysteine, 10% porcine follicular fluid, 1 mM dibutyryl cyclic adenosine monophosphate, and 0.1 IU/mL human menopausal gonadotrophin for 20 h and then culture without dbcAMP and hMG for another 18 to 24 h. Fetal cells were isolated from a male fetus and two female fetuses, and cultured in ES-DMEM medium containing 10% FCS. Enucleated oocytes were fused with fetal fibroblasts (passage 4 to 15). Reconstructed embryos were cultured in NCSU-23 with 4 mg/mL BSA under mineral oil at 39°C in 5% CO2 in air for up to 6 days. NT eggs that had been activated with electric pulses and cultured for 1 or 2 days were transported to the experimental station in modified NCSU-23 with antibiotics. NT embryos were surgically transferred into the oviducts of recipients between Day 27 and Day 30; pregnancy was determined by ultrasound. The potential of NT embryos to develop into blastocysts was not different among donor cells of different origins. However, the mean cell number of in vivo female and male blastocysts (83.8 ± 46.2 to 99.2 ± 55.7) was higher than in in vitro culture of NT groups (31.4 ± 8.29 to 33.2 ± 10.15). A total of 11,535 NT embryos (1- to 8-cell stage) were surgically transferred into 66 surrogate gilts. Among fourteen pregnant gilts, four recipients aborted during the period of conception. Five pregnant gilts delivered fifteen female piglets, 1.28 ± 0.33 kg (0.48∼1.83 kg) in female piglets and 0.84±0.25 kg (0.45∼1.25 kg) in male piglets. Nine live cloned female (60.0%) and four male piglets (18.2%) were produced. According to these results, survival rates and birth weights of female cloned piglets were higher than those of cloned male piglets (P < 0.05). This study suggests that use of female, compared with male, fetal fibroblast cells as nuclear donors may increase cloning outcomes. This work was supported in part by a grant program from RDA(Biogreen21) and Cho-A, Republic of Korea.

scholarly journals 75 RABBIT NUCLEAR TRANSFER WITH CULTURED SOMATIC CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 187 ◽  
Author(s):  
F. Yang ◽  
B. Kessler ◽  
S. Ewerling ◽  
E. Wolf ◽  
V. Zakhartchenko
Keyword(s):  
Nuclear Transfer ◽  
Cultured Cells ◽  
Somatic Cells ◽  
Cumulus Cells ◽  
Cell Stage ◽  
Electric Pulses ◽  
Donor Cells ◽  
Fetal Fibroblasts

Cloned rabbits have been obtained by somatic cell nuclear transfer (SCNT) only with fresh, non-cultured cumulus cells (Chesne et al. 2002 Nat. Biotechnol. 20, 366–369). For the purpose of generating transgenic animals by SCNT, donor cells must be cultured and modified prior to use as nuclear donors. The objective of this study was to optimize the SCNT procedure using cultured cumulus or fibroblast cells. MII oocytes were harvested from superovulated Zika rabbits, and maternal chromosomes were removed by demecolcine-assisted enucleation (Yin et al. 2002 Biol. Reprod. 67, 442–446). Two types of somatic cells originating from Ali/Bass rabbits were used as nuclear donors: cumulus cells collected from in vivo-matured oocytes and cultured for 1–5 passages, and primary fetal fibroblasts obtained from Day 16 fetuses and grown to confluence or starved for 4–5 days. Somatic donor cells and recipient cytoplasts were fused with 2 electric pulses (1.95 kV/cm, 25 μs each, 1 s interval). Twenty to 40 min after fusion, cloned embryos were activated first with the same electropulses as for fusion, and then immediately followed by 1 h incubation in 2 mM 6-dimethylaminopurine and 5 μg/mL cytochalasin B in culture medium (B2 medium supplemented with 10% FCS). Cloned embryos were either transferred at the 2- and 4-cell stage to asynchronized recipients or cultured in vitro for 6 days. Data were compared using chi-square test, and differences were considered significant when P < 0.05. Our results demonstrate that cloned rabbits can be produced by SCNT with cultured cells but the efficiency of this technique is still very low irrespective of the type of donor cells. Table 1. Development of cloned embryos derived from somatic cells This research was supported by the Therapeutic Human Polyclonals, Inc.

59 HIGHER BLASTOCYST FORMATION OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DOES NOT GUARANTEE BETTER PREGNANCY IN PIG

2007 ◽  
Vol 19 (1) ◽  
pp. 147
Author(s):  
E. Lee ◽  
K. Song ◽  
Y. Jeong ◽  
S. Hyun
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Skin Fibroblasts ◽  
Miniature Pig ◽  
Donor Cells ◽  
Fetal Fibroblasts

Generally, blastocyst (BL) formation and embryo cell number are used as main parameters to evaluate the viability and quality of in vitro-produced somatic cell nuclear transfer (SCNT) embryos. We investigated whether in vitro development of SCNT pig embryos correlates with in vivo viability after transfer to surrogates. For SCNT, cumulus–oocyte complexes (COCs) were matured in TCM-199 supplemented with follicular fluid, hormones, EGF, cysteine, and insulin for the first 22 h and in a hormone-free medium for 18 h. Three sources of pig skin cells were used as nuclear donor: (1) skin fibroblasts of a cloned piglet that were produced by SCNT of fetal fibroblasts from a Landrace × Yorkshire × Duroc F1 hybrid (LYD), (2) skin fibroblasts of a miniature pig having the human decay accelerating factor gene (hDAF-MP), and (3) skin fibroblasts of a miniature pig with a different strain (MP). MII oocytes were enucleated, subjected to nuclear transfer from a donor cell, electrically fused, and activated 1 h after fusion. SCNT embryos were cultured in a modified NCSU-23 (Park Y et al. 2005 Zygote 13, 269–275) for 6 days or surgically transferred (110–150 fused embryos) into the oviduct of a surrogate that showed standing estrus on the same day as SCNT. Embryos were examined for cleavage and BL formation on Days 2 and 6, respectively (Day 0 = the day of SCNT). BLs were examined for their cell number after staining with Hoechst 33342. Pregnancy was diagnosed by ultrasound 30 and 60 days after embryo transfer. Embryo cleavage was not affected by donor cells (82, 81, and 72% for LYD, hDAF-MP, and MP, respectively), but BL formation was higher (P &lt; 0.05) in hDAF-MP (16%) than in LYD (9%) and MP (6%). MP showed higher (P &lt; 0.05) BL cell number (46 cells/BL) than hDAF-MP (34 cells) but did not show a difference from LYD (37 cells). LYD and MP showed higher pregnancy rates (Table 1) on Days 30 and 60, even though they showed lower BL formation in vitro. Due to a relatively small number of embryo transfers through a limited period, we could not exclude any possible effects by seasonal or operational differences. These results indicated that pregnancy did not correlate with in vitro BL formation of SCNT pig embryos but rather were affected by the source of donor cells. Table 1.In vivo development of somatic cell nuclear transfer pig embryos derived from different sources of donor cells This work was supported by the Research Project on the Production of Bio-organs (No. 200506020601), Ministry of Agriculture and Forestry, Republic of Korea.

102 RABBIT CLONING: HISTONE ACETYLATION STATUS OF DONOR CELLS AND CLONED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 168
Author(s):  
V. Zakhartchenko ◽  
F. Yang ◽  
R. Hao ◽  
E. Wolf
Keyword(s):  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Epigenetic Mark ◽  
Cloning Efficiency ◽  
Developmental Potential ◽  
Acetylation Status ◽  
Donor Cells ◽  
Fetal Fibroblasts

Epigenetic status of the genome of a donor nucleus is likely to be associated with the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). Prevention of epigenetic errors by manipulation of the epigenetic status of donor cells is expected to result in improvement of cloning efficiency. In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Ali/Bas) into metaphase II (MII) oocytes and analyzed the levels of histone H3K9 acetylation in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with one or two blastomeres from in vitro-fertilized or parthenogenetic embryos. Histone acetylation in donor cells and cloned embryos was detected by anti-acH3K9 antibody using Western immunoblot analysis or immunochemistry, respectively. Data were analyzed by chi-square (developmental rates) or Student-Newman-Keuls (histone acetylation) test. The levels of acetylated histone H3K9 were higher in RCCs than in RFFs (P &lt; 0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC-cloned embryos induced a higher initial pregnancy rate as compared to RFF-cloned embryos (40% vs. 20%; P &lt; 0.05). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed; a live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly (P &lt; 0.05) increased the level of histone H3K9/14 acetylation and the proportion of nuclear transfer embryos developing to blastocyst (49% vs. 33% with non-treated RFF; P &lt; 0.05). The distribution of signals for acH3K9 in either group of cloned embryos did not resemble that in in vivo-fertilized embryos, suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo-derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and can be a useful epigenetic mark to predict efficiency of SCNT rabbits. This work was supported by the Bayerische Forschungsstiftung and by Therapeutic Human Polyclonals, Inc.

282 IMPROVED QUALITY OF PORCINE BLASTOCYSTS BY AGGREGATION OF EMBRYOS AT THE 4 - 8-CELL STAGE

2006 ◽  
Vol 18 (2) ◽  
pp. 248
Author(s):  
S.-G. Lee ◽  
C.-H. Park ◽  
D.-H. Choi ◽  
H.-Y. Son ◽  
C.-K. Lee
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Transgenic Animals ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Vitro Fertilization

Use of blastocysts produced in vitro would be an efficient way to generate embryonic stem (ES) cells for the production of transgenic animals and the study of developmental gene regulation. In pigs, the morphology and cell number of in vitro-produced blastocysts are inferior to these parameters in their in vivo counterparts. Therefore, establishment of ES cells from blastocysts produced in vitro might be hindered by poor embryo quality. The objective of this study was to increase the cell number of blastocysts derived by aggregating 4–8-cell stage porcine embryos produced in vitro. Cumulus–oocyte complexes were collected from prepubertal gilt ovaries, and matured in vitro. Embryos at the 4–8-cell stage were produced by culturing embryos for two days after in vitro fertilization (IVF). After removal of the zona pellucida with acid Tyrode’s solution, one (1X), two (2X), and three (3X) 4–8-cell stage embryos were aggregated by co-culturing them in aggregation plates followed by culturing to the blastocyst stage. After 7 days, the developmental ability and the number of cells in aggregated embryos were determined by staining with Hoechst 33342 and propidium iodide. The percentage of blastocysts was higher in both 2X and 3X aggregated embryos compared to that of 1X and that of intact controls (Table 1). The cell number of blastocysts also increased in aggregated embryos compared to that of non-aggregated (1X) embryos and controls. This result suggests that aggregation might improve the quality of in vitro-fertilized porcine blastocysts by increasing cell numbers, thus becoming a useful resource for isolation and establishment of porcine ES cells. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the pluripotent cell population by staining for Oct-4 and to apply improved aggregation methods in nuclear-transferred (NT) porcine embryos. Table 1. Development, cell number, and ICM ratio of aggregated porcine embryos

98 SUCCESSFUL PREGNANCIES FOLLOWING TRANSFER OF VITRIFIED PORCINE EMBRYOS DERIVED FROM IN VITRO-MATURED OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 157 ◽  
Author(s):  
K. Hiruma ◽  
H. Ueda ◽  
H. Saito ◽  
C. Tanaka ◽  
N. Maeda ◽  
...  
Keyword(s):  
Calf Serum ◽  
Developmental Competence ◽  
Penicillin G ◽  
Cell Stage ◽  
Epidermal Growth ◽  
Potassium Penicillin ◽  
The One ◽  
Parthenogenetic Embryos

To date only in vivo-produced embryos have successfully produced live piglets after cryopreservation. In this study, we aimed to produce piglets from vitrified embryos derived from in vitro matured (IVM) oocytes. Cumulus-oocyte complexes collected from ovaries obtained at a local slaughterhouse were matured for 44 to 45 h in NCSU23 MEDIUM supplemented with 0.6 mM cysteine, 10 ng/mL epidermal growth factor, 10% (v/v) porcine follicular fluid, 75 �g/mL potassium penicillin G, 50 �g/mL streptomycin sulfate, and 10 IU/mL eCG/ hCG. These IVM oocytes were either activated for parthenogenesis or in vitro-fertilized (IVF). For IVF, oocytes were incubated with 5 � 106/mL of cryopreserved epididymal sperm in PGM-tac medium (Yoshioka et al. 2003 Biol. Reprod. 69, 2092-2099) for 20 h. Embryos were treated for removal of cytoplasmic lipid droplets (delipation; Nagashima et al. 1995 Nature 374, 416) at the 4- to 8-cell stages, around 50 to 54 h after activation or insemination. After culture in NCSU23 for 15 h, they were vitrified by the minimum volume cooling (MVC) method. Embryos were equilibrated with equilibration solution containing 7.5% (v/v) ethylene glycol (EG), 7.5% (v/v) dimethylsulfoxide (DMSO), and 20% (v/v) calf serum for 4 min, followed by exposure to vitrification solution containing 15% EG, 15% DMSO, 0.5 M sucrose, and 20% calf serum. Embryos were then loaded onto a Cryotop (Kitazato Supply Co., Tokyo, Japan) and immediately plunged into liquid nitrogen. Vitrified embryos were examined for viability in vitro and in vivo after warming. Their in vitro developmental competence was compared to that of corresponding control (nonvitrified) embryos. Vitrified 4- to 8-cell stage embryos, both parthenogenetic and IVF, showed developmental competence into blastocysts comparable to that of control embryos (parthenogenetic: 46.8%, 36/77 vs. 51.7%, 31/60; IVF: 40.0%, 30/75 vs. 44.3%, 35/79). Of four surrogate gilts that received a total of 251 vitrified parthenogenetic embryos, three became pregnant and had 20 fetuses (8.0%, 22 to 23 days old). Three surrogates gilts that received 267 vitrified IVF embryos all became pregnant. Of those, the one that received 47 embryos was confirmed to have eight fetuses (17.0%, 22 days old) by autopsy. The other two were examined by ultrasonography at 56 and 95 days of gestation and found to be pregnant. These results suggest that porcine embryos derived from IVM oocytes have a potential to develop into live offspring after delipation and MVC vitrification. This study was supported by PROBRAIN.

43 COMPARISON OF THE EFFECTS OF PRE-TREATMENT WITH SODIUM CHLORIDE, SUCROSE AND TREHALOSE ON DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 121
Author(s):  
L. Lin ◽  
P. Kragh ◽  
S. Purup ◽  
Y. Du ◽  
X. Zhang ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
Nuclear Transfer ◽  
Technical Assistance ◽  
Recovery Period ◽  
Cell Number ◽  
Developmental Competence ◽  
Fetal Fibroblasts ◽  
Pre Treatment ◽  
Maximum Humidity

Modified environmental stress was reported to improve the developmental competence and cryotolerance of porcine oocytes, such as high hydrostatic pressure (HHP; Du et al. 2008 Cloning Stem Cells, Epub ahead of print) and osmotic stress (Lin et al. 2008 Reprod. Biomed. Online, in press). HHP also improved the cryotolerance of bovine and murine blastocysts (Pribenszky et al. 2005a Reprod. Dom. Anim. 40, 338–344; Pribenszky et al. 2005b Anim. Reprod. Sci. 87, 143–150). In the present study we compared the effects of NaCl with that of concentrated solutions of two non-permeable osmotic agents, sucrose and trehalose on in vitro maturated oocytes. A total of 2050 slaughterhouse-derived porcine cumulus–oocyte complexes (COCs) were matured for 41–42 h, and then put into 800 μL T2 (HEPES-buffered TCM-199 [Earle’s salts] with 2% cattle serum) supplemented with additional NaCl, sucrose or trehalose with the same osmotic level (588 mOsmol) in 4-well dishes and incubated for 1 h at 38.5°C in air. COCs incubated in T2 under the same conditions without supplementation were used as controls. Subsequently COCs were incubated in IVM medium for 1 h at 38.5°C in 5% CO2 with maximum humidity. After this recovery period cumulus cells were removed with 1 mg mL–1 hyaluronidase and pipetting, and oocytes were used as recipients for somatic nuclear transfer with handmade cloning (HMC) method. Porcine fetal fibroblasts were used as nuclear donor cells. Embryo culture was performed in PZM-3 medium (Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) in 5% CO2, 5% O2 and 90% N2 and maximum humidity. Cleavage and blastocyst rates were checked on Day 1 and Day 6, respectively. Cell numbers were counted after fixation in glycerol containing 20 μg mL–1 Hoechst 33342 fluorochrome on Day 6. t-test was performed for statistical calculations with SPSS 11.0 program (SPSS, Chicago, IL, USA). Results are shown in Table 1. Osmotic stress with both permeable and non-permeable agents increased developmental competence of porcine IVM oocytes. NaCl seems to be more appropriate for the purpose, as the other two components resulted in decreased cell number in blastocysts after somatic cell nuclear transfer (SCNT). In conclusion, a simple NaCl pre-treatment of oocytes has improved the in vitro efficiency of porcine SCNT. Table 1.Developmental competence of porcine HMC embryos derived from oocytes treated with different agents The authors thank Ruth Kristensen, Anette Pedersen, Janne Adamsen and Klaus Villemoes for their help and excellent technical assistance.

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.

159 EFFECT OF 3-ISOBUTYL-1-METHYLXANTHINE (IBMX) ON THE GERMINAL VESICLE STAGE OF PORCINE OOCYTES DURING OOCYTE COLLECTION IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 193
Author(s):  
R. Appeltant ◽  
J. Beek ◽  
D. Maes ◽  
A. Van Soom
Keyword(s):  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Guanosine Monophosphate ◽  
Developmental Competence ◽  
Meiotic Arrest ◽  
Oocyte Collection

When using modern maturation conditions for in vitro maturation, pig oocytes yield ~20% blastocysts only. One problem is that cumulus cells, which are normally connected with the immature oocyte by cellular projections penetrating through the zona pellucida and with the oolemma via gap junctions, are prematurely losing these connections after the cumulus–oocyte complex is removed from the follicle. The oocyte possesses a type 3 phosphodiesterase, which degrades 3′,5′-cyclic adenosine monophosphate (cAMP), and this activity is inhibited by supply of 3′,5′-cyclic guanosine monophosphate (cGMP) to the oocyte via the cumulus cells. Consequently, cAMP levels, which are typically high during early stages of oocyte maturation in vivo, decrease, leading to spontaneous nuclear maturation and oocytes of low developmental competence. Therefore, the maintenance of these cumulus-oocyte connections is important to keep cAMP high and the oocyte under meiotic arrest. One way to prevent this drop in cAMP is using N6, 2′-o-dibutyryladenosine 3′,5′-cyclic monophosphate sodium (dbcAMP) that causes an arrest at germinal vesicle (GV) stage II (Funahashi et al. 1997 Biol. Reprod. 57, 49–53). Another option is collecting the oocytes in a medium containing the phoshodiesterase inhibitor, IBMX. The present study investigated the influence of IBMX on the progression of the GV of the oocyte after collection, just before the start of the maturation procedure. The GV stage was defined according to Sun et al. (2004 Mol. Reprod. Dev. 69, 228–234). In parallel with the findings on dbcAMP, we hypothesised an arrest at GV II by the presence of IBMX during collection. One group of oocytes were collected in HEPES-buffered TALP without IBMX (n = 375) and another group in the same medium containing 0.5 mM IBMX (n = 586). An average incubation time of 140 min was applied in both groups, and 3 replicates were performed. The proportions of oocytes before or at GV II and beyond GV II were compared in both groups using logistic regression analysis. The proportion of oocytes was included as dependent variable and group (IBMX addition or not) as independent variable. Replicate was also included in the model. The proportion of oocytes before or at GV II was not statistically significant between the group without and the group with IBMX (59.2 v. 58.7% respectively; P > 0.05). In conclusion, the use of IBMX during oocyte collection did not influence the state of the germinal vesicle of the oocyte during collection, indicating that IBMX did not cause a meiotic arrest in the oocytes during collecting in vitro.

scholarly journals 152EFFECT OF REPLACEMENT OF PYRUVATE/LACTATE IN CULTURE MEDIUM WITH GLUCOSE ON PREIMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 198
Author(s):  
N.W.K. Karja ◽  
S. Medvedev ◽  
D. Fuchimoto ◽  
A. Onishi ◽  
M. Iwamoto ◽  
...  
Keyword(s):  
Energy Source ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Culture Period ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Energy Substrates ◽  
Anova Test

Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) reported that replacement of pyruvate and lactate with glucose, as energy substrates, at 48h of culture in IVC medium enhanced the quality of IVP porcine blastocysts. However, the exact time during early cleavage stages when the utilization of glucose as an energy source is optimal has not yet been determined. The purpose of this study was to examine the effects of glucose supplementation at different times of culture on the developmental competence of IVP porcine embryos. Porcine cumulus-oocytes complexes were matured in modified NCSU-37 solution and fertilized in vitro according to Kikuchi et al. All cultures were performed at 38.5°C, 5% O2, 5% CO2, and 90% N2. In experiment 1, after being fertilized (Day 0), putative zygotes (1158 in 6 trials) were cultured in NCSU-37 supplemented with 0.4% BSA, 0.17mM sodium pyruvate, and 2.73mM sodium lactate (IVC-pyr/lac). Embryos (30–50 in each group) were then transferred into NCSU-37 supplemented with 0.4% BSA and 5.55mM D-glucose (IVC-glu) at 24, 48, 72, 96, or 118h of culture. As control groups, putative zygotes (391) were cultured in IVC-pyr/lac or IVC-glu for the whole culture period. In experiment 2, after being fertilized, putative zygotes (543 in 4 trials, 30–50 in each group) were cultured in IVC-pyr/lac, and then were transferred into IVC-glu at 48h, 53h, 58h, or 63h of culture, because glycolytic activity of in vitro-derived porcine embryos was reported to increase around the 8-cell stage, and some embryos develop to that stage before 72h of culture in experiment 1. All embryos were cultured for 6 days, and then development to the blastocyst stage and number of cells per blastocyst were assessed. When IVF embryos were cultured in IVC pyr/lac for 24h or 48h and subsequently in IVC-glu until day 6 in experiment 1, the rates of blastocyst formation were significantly higher (P&lt;0.05, ANOVA test) than those of embryos cultured in IVC-pyr/lac for the whole culture period (24.4% and 23.0% v. 14.5%, respectively). However, when IVC pyr/lac was replaced with IVC-glu, there were no significant differences between the energy source replacement groups and the glucose-only group in terms of the proportions of cleavage, development to the blastocyst stage and mean cell number per blastocyst (P&gt;0.05, ANOVA test) (15.2%–24.4%, and 16.8%, respectively). Replacement of pyruvate and lactate with glucose at 58h of culture in experiment 2 significantly enhanced the rate of blastocyst formation (P&lt;0.05, ANOVA test) but not the mean cell number compared with zygotes in which the replacement was done at 48, 53, and 63h of culture (31.3% v. 20.6%, 20.8%, and 21.1%, respectively) (P&lt;0.05, ANOVA test). In conclusion, replacement of pyruvate and lactate with glucose as energy substrates was optimal at 58h of culture for the in vitro development of pig embryos to the blastocyst stage.

81 HIGH THROUGHPUT VITRIFICATION OF IN VIVO FERTILIZED AND IN VITRO CULTURED PORCINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 146
Author(s):  
C. N. Murphy ◽  
L. D. Spate ◽  
B. K. Bauer ◽  
R. S. Prather
Keyword(s):  
Ethylene Glycol ◽  
Uv Light ◽  
Cell Number ◽  
Total Cell ◽  
Swine Industry ◽  
Total Cell Number ◽  
Cell Stage ◽  
High Osmolarity

One barrier to successfully making embryo transfer viable in the swine industry is an inability to consistently cryopreserve oocytes and embryos. This process is made difficult by the high lipid content of porcine oocytes and embryos. The objective of this study was to test the in vivo fertilized embryo’s sensitivity to vitrification. Gilts were inseminated on the first day of standing oestrus (Day 0) and then again 12 h later. On Day 2 the oviducts and tip of the uterine horns were flushed with PVA-treated TL-HEPES and 2-cell stage embryos were collected and placed into PVA-treated TL-HEPES and centrifuged at 17 000 × g. The treatment groups were 1) 300 mOsmo centrifuged for 6 min, 2) 500 mOsmo centrifuged for 6 min, 3) 500 mOsmo centrifuged for 12 min, and 4) 500 mOsmo centrifuged for 18 min. After centrifugation the embryos were transferred to Porcine Zygote Medium 3 (PZM3) and cultured to Day 6 or 7 at which point blastocysts were vitrified using 10% DMSO, 10% ethylene glycol in M199 supplemented with 20% FBS (holding medium) for 2 min. Embryos were transferred to holding media with 20% DMSO and 20% ethylene glycol and drawn into an open pulled straw via capillary reaction; it was then submerged into LN2. Embryos were thawed using a step down concentration of 0.33 mM and then 0.2 mM sucrose in holding media each for 6–7 min and then were moved to holding medium alone for 6 to 7 min. The embryos were washed in PZM3, then transferred to 500 μL of PZM3 and cultured for 18 h. Re-expanded embryos were observed, and the nuclei of all embryos were stained with Biz-benzimide and visualised with UV light to determine total cell number. After the embryos were centrifuged and cultured, there was no difference in development to blastocyst (SAS Institute, Cary, NC, USA; Proc GLM) with a mean percentage blastocyst of 85.1% and an N of 54, 51, 53, and 51, respectively, for each treatment. After thawing, percentage of embryos re-expanded was 23.5a, 26.4a,b, 43.2a,b, and 45.6b, respectively. Data was analysed using a PROC GLM in SAS (P < 0.05), with 37, 43, 30, and 36 embryos in each group, respectively. No difference in total cell number across treatments was detected after analysis using PROC GLM in SAS (P < 0.05) with a mean cell number of 29.0. These data suggest that in vivo matured and fertilized blastocysts can survive high osmolarity treatment, centrifugation, and vitrification. The data also show that a high osmolarity treatment centrifuged for 18 min leads to a greater number of re-expanded embryos post-thaw, which may be attributed to better separation of the lipid. Funded by the NIH NCRR R21RR025879 and Food for the 21st Century.

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