Effects of the time interval between fusion and activation on in vitro rabbit nuclear transfer efficiency when nuclear donor cells are derived from older adults

Zygote ◽  
2004 ◽  
Vol 12 (2) ◽  
pp. 133-141 ◽  
Author(s):  
R.P. Cervera ◽  
F. Garcia-Ximénez
Keyword(s):  
Older Adult ◽  
Exposure Time ◽  
Blastocyst Stage ◽  
Adult Rabbit ◽  
Time Interval ◽  
Unfavourable Effect ◽  
Donor Cells ◽  
Hatching Rates

Cloning older adult rabbits can serve as a model in animal breeding, biodiversity preservation and in human therapeutic cloning. To establish the required exposure time of fibroblasts from these kind of animals to reprogramming factors, in the present study three different time intervals between fusion and activation were tested (30 min, 30-ADF group; 60 min, 60-ADF group; and 90 min, 90-ADF group). Vitrified epithelial fibroblasts derived from four older adult rabbit females (D1, D2, D3 and D4) and cultured from passages 0 to 4 were used as nuclear donors. Nuclear status of reconstructed embryos was not evaluated. No differences were observed in blastocyst rate (30-ADF 21% vs 60-ADF 19% vs 90-ADF 18%). Differences in hatching rates did not reach significance (30-ADF 11% vs 60-ADF 18% vs 90-ADF 18%). However, in the 60- and 90-ADF groups, embryos reached the blastocyst stage earlier than in the 30-ADF group (day 4: 40% and 50% vs 8%; p>0.05). Moreover, the quality of blastocysts (good vs poor) was lower in the 30-ADF group (good: 30-ADF 38% vs 60-ADF 90% vs 90-ADF 90%; p>0.05). Overall, these results suggest an unfavourable effect of the shortest exposure time tested (30 min). Differences between specimen origins were detected (blastocyst and hatching rates: D2 (26%; 25%) and D4 (25%; 27%) vs D1 (10%; 11%) and D3 (12%; 12%)), but significance were not reached. Effect of culture passage was not detected in any parameter studied.

Overexpression of MicroRNA-29b Decreases Expression of DNA Methyltransferases and Improves Quality of the Blastocysts Derived from Somatic Cell Nuclear Transfer in Cattle

2018 ◽  
Vol 24 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Shuang Liang ◽  
Zheng-Wen Nie ◽  
Jing Guo ◽  
Ying-Jie Niu ◽  
Kyung-Tae Shin ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cellular Proliferation ◽  
Dna Methyltransferases ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Somatic Cell Reprogramming

AbstractMicroRNA (miR)-29b plays a crucial role during somatic cell reprogramming. The aim of the current study was to explore the effects of miR-29b on the developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos, as well as the underlying mechanisms of action. The expression level of miR-29b was lower in bovine SCNT embryos at the pronuclear, 8-cell, and blastocyst stages compared within vitrofertilized embryos. In addition, miR-29b regulates the expression of DNA methyltransferases (Dnmt3a/3bandDnmt1) in bovine SCNT embryos. We further investigated SCNT embryo developmental competence and found that miR-29b overexpression during bovine SCNT embryonic development does not improve developmental potency and downregulation inhibits developmental potency. Nevertheless, the quality of bovine SCNT embryos at the blastocyst stage improved significantly. The expression of pluripotency factors and cellular proliferation were significantly higher in blastocysts from the miR-29b overexpression group than the control and downregulation groups. In addition, outgrowth potential in blastocysts after miR-29b overexpression was also significantly greater in the miR-29b overexpression group than in the control and downregulation groups. Taken together, these results demonstrated that miR-29b plays an important role in bovine SCNT embryo development.

155 QUALITY OF PORCINE EMBRYO PRODUCED IN TWO EMBRYO CULTURE MEDIA AS ASSESSED BY TOTAL CELL NUMBER AND TIME COURSE OF BLASTOCYST HATCHING

2006 ◽  
Vol 18 (2) ◽  
pp. 185 ◽  
Author(s):  
Y. Agca ◽  
H. Men ◽  
S. F. Mullen ◽  
L. K. Riley ◽  
R. S. Prather ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Time Course ◽  
Culture Media ◽  
Cell Number ◽  
Total Cell ◽  
Total Cell Number ◽  
Blastocyst Hatching ◽  
Hatching Rates

The ability to produce porcine embryos of good quality will have a significant impact on a number of porcine assisted reproductive technologies, such as cloning, intracytoplasmic sperm injection, and embryo cryopreservation. However, porcine embryos resulting from current serum-free embryo culture systems differ significantly both structurally and functionally from those derived in vivo (Wang et al. 1999 Mol. Reprod. Dev. 53, 99-107). In this experiment, the quality of porcine embryos produced by North Carolina State University (NCSU)-23 medium (Petters and Wells 1993 J. Reprod. Fertil. Suppl. 1993, 48, 61-73) and porcine zygote medium (PZM)-1 (Yoshioka et al. 2002 Biol. Reprod. 66, 112-119) were compared by assessing the total cell number and the time course of in vitro blastocyst hatching. Porcine embryos were produced by in vitro maturation and fertilization using serum-free systems. After fertilization, presumptive zygotes were randomly allocated to either PZM-1 or NCSU-23 for subsequent development. On Day 4 of culture, the embryo culture media were supplemented with 10% fetal bovine serum (FBS). Day 6 blastocysts from each group were counted and the blastocysts were subsequently fixed in 4% formalin for counting the total cell number. The cell number in each embryo was determined by counting the nuclei after staining with bisbenzimide (Hoechst 33342). To assess the hatching ability of blastocysts, Day 6 blastocysts were cultured until Day 9 and hatched blastocysts were counted daily. Day 6 blastocyst rates (ratio of blastocysts to oocytes) and total cell number count were replicated three times. The time course of blastocyst hatching experiment was repeated four times. The data were analyzed using a chi-square test, Fisher's exact test, or Student's t-test. The blastocyst rate from culture in PZM-3 was 19.4 � 0.96% (mean � SEM), which was similar to that (16.7 � 3.2%) resulting from culture in NCSU-23 (P > 0.05). However, the total cell number in Day 6 blastocysts cultured in PZM-3 was significantly higher than for blastocysts cultured in NCSU-23 (57 � 3.1 vs. 46 � 1.7; P < 0.01). The total hatching rates (ratio of hatched blastocysts to total blastocysts) by Day 9 were similar between the two culture systems (50.1 � 9.1% vs. 50.7 � 4.1%; P > 0.05). However, on Day 6, 2.1% of blastocysts from PZM-3 culture hatched whereas no blastocysts from NCSU-23 culture hatched. The cumulative hatching rates from PZM-3 culture on Day 7 were significantly higher than those from NCSU-23 culture (15.1 � 3.8% vs. 2.6 � 1.1%; P < 0.01). In conclusion, these data suggest that blastocysts produced in PZM-3 medium have better quality than blastocysts produced in the NCSU-23 culture system as assessed by the total cell number and the time course of blastocyst hatching. This project was supported by a grant from the National Institutes of Health (U42 RR 018877).

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

42 DEVELOPMENT OF PIG EMBRYOS CLONED FROM DONOR CELLS TREATED WITH TRICHOSTATIN A

2008 ◽  
Vol 20 (1) ◽  
pp. 101 ◽  
Author(s):  
J. Li ◽  
Y. Du ◽  
P. M. Kragh ◽  
S. Purup ◽  
K. Villemoes ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Calcium Ionophore ◽  
Blastocyst Stage ◽  
Fibroblast Cells ◽  
Embryonic Genome ◽  
Donor Cells ◽  
Deacetylase Inhibitor ◽  
Vitro Development

Development to the blastocyst stage following nuclear transfer is dependent on the donor cell's ability to reprogram its genome to a totipotent state. Reprogramming of the transferred somatic nuclei must be completed by the time normal activation of the embryonic genome occurs (Solter 2000 Nat. Rev. Genet. 1, 199–207). Recently, Enright et al. (2003 Biol. Reprod. 69, 896–901) reported that in vitro development of cloned cow embryos was improved by treatment of donor cells with a histone deacetylase inhibitor, TrichostatinA (TSA). So far, there are no reports available for adult pig fibroblast cells treated with TSA. The objective of this study was to investigate whether the development of handmade cloned embryos in pig could be improved by using TSA-treated donor cells. Adult pig fibroblast cells were treated with 100, 150, or 200 nm TSA for 24 h, compared to untreated controls, and were then used as donor cells. The cells were electrofused with handmade enucleated pig oocytes separately and were activated with calcium ionophore and cycloheximide. They were subsequently cultured in porcine zygote medium 3 (PZM-3; Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) using the well of the well system (WOW; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264). Experiments were repeated 4 times and the data were analyzed with AVEDEV and t-test in Excel (Microsoft Excel 2007). The cleavage rates and the total cell numbers per blastocyst were similar between groups (P > 0.05), as shown in Table 1. However, the cloned blastocyst rate using donor cells treated with 100 nm TSA was higher than in the other groups (69.9 ± 4.7% v. 43.6 ± 4.3%, 43.1 ± 5.8%, or 46.6 ± 3.6%; P < 0.05), as shown in Table 1. These data suggest that proper TSA treatment for donor cells before somatic cloning improves the rate of development of porcine handmade cloned embryos to the blastocyst stage. Further research is needed to examine the in vivo development of embryos reconstructed with TSA-treated donor cells. Table 1. Developmental ability of cloned pig embryos derived fromTSA-treated donor cells

144 USE OF A NOVEL BOVINE EMBRYO CULTURE MEDIUM TO IMPROVE BLASTOCYST DEVELOPMENT AND SURVIVAL FOLLOWING VITRIFICATION

2010 ◽  
Vol 22 (1) ◽  
pp. 231
Author(s):  
J. Block ◽  
L. Bonilla ◽  
P. J. Hansen
Keyword(s):  
Amino Acids ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Fresh Embryos ◽  
Hatching Rates

The objective of the present study was to determine whether culture of bovine embryos in a proprietary serum-free culture medium, Block-Bonilla-Hansen-7 (BBH-7), could improve development to the blastocyst stage and enhance survival following vitrification. For Exp. 1, embryos were produced in vitro and cultured in BBH-7 or modified synthetic oviductal fluid (mSOF; as in zygote 10:341 except with 10 μL mL-1 of nonessential amino acids, 20 μL mL-1 of essential amino acids, and 1 mg mL-1 of polyvinyl alcohol instead of albumin) in 5% (v/v) oxygen. Grade 1 expanded blastocysts were harvested at Day 7 post-insemination and vitrified using the open-pulled straw method (Vagta et al. 1998 Mol. Reprod. Dev. 51, 53-58). Vitrified embryos were thawed and cultured in vitro in either mSOF or BBH-7 supplemented with 10% fetal bovine serum and 50 μM dithiolthreitol. Re-expansion and hatching rates were recorded at 24, 48, and 72 h post-thaw. There was no effect of culture medium on cleavage rate. The proportion of oocytes that developed to the blastocyst and advanced blastocyst stages (expanded, hatching, and hatched) at Day 7 was higher (P < 0.001) for embryos cultured in BBH-7 than for embryos cultured in mSOF (41.9 ± 2.0 v. 14.7 ± 2.0% and 31.1 ± 1.3 v. 6.4 ± 1.3%, respectively). There was no effect of culture medium on re-expansion rates at 24, 48, and 72 h post-thaw or on hatching rates at 48 or 72 h. However, the proportion of embryos that were hatching or had hatched by 24 h post-thaw was higher (P < 0.001) for BBH-7 than for mSOF (59.0 ± 0.5 v. 26.7 ± 0.5%). For Exp. 2, late lactation and/or repeat breeder, lactating Holstein cows were synchronized for timed embryo transfer using the OvSynch-56 protocol. Embryos were produced in vitro and cultured in BBH-7 in 5% (v/v) oxygen. Vitrified embryos were produced as for Exp. 1. Fresh embryos were grade 1 expanded blastocysts harvested at Day 7 after insemination. A single embryo was transferred at Day 7 after putative ovulation to all cows with a corpus luteum confirmed by ultrasonography. Pregnancy was diagnosed at Day 28-30 of gestation by ultrasonography. There was no difference in the proportion of recipients that became pregnant after receiving either a fresh (7/18 = 39%) or vitrified (10/27 = 37%) embryo cultured in BBH-7. The results of the present study indicate that BBH-7 can be used to increase the proportion of oocytes that develop to the blastocyst stage. Moreover, the results demonstrate that vitrified embryos produced after culture in BBH-7 can achieve pregnancy rates similar to those obtained using fresh embryos. Support: USDA 2006-55203-17390 and Southeast Milk Checkoff Program.

34 EFFECTS OF L-CARNITINE AND trans-10,cis-12 CONJUGATED LINOLEIC ACID SUPPLEMENTATION DURING MATURATION ON DEVELOPMENT AND CRYOTOLERANCE OF BOVINE EMBRYOS PRODUCED IN VITRO

2016 ◽  
Vol 28 (2) ◽  
pp. 147
Author(s):  
J. Block ◽  
A. M. Zolini ◽  
E. Carrascal-Triana ◽  
A. Ruiz ◽  
P. J. Hansen ◽  
...  
Keyword(s):  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Sodium Pyruvate ◽  
Maturation Medium ◽  
Fetal Bovine ◽  
Hatching Rates

The objective of the present study was to determine the effect of supplementation of maturation media with L-carnitine and trans-10,cis-12 conjugated linoleic acid (CLA) on embryo development and survival following cryopreservation. Immature bovine cumulus-oocyte complexes (n = 1796) were harvested from abattoir-derived ovaries and randomly assigned in a 2 × 2 factorial design to be matured in maturation medium [TCM-199 with Earle salts supplemented with 10% (vol/vol) bovine steer serum, 2 μg mL–1 oestradiol 17-β, 20 μg mL–1 bovine FSH, 22 μg mL–1 sodium pyruvate, 50 μg mL–1 gentamicin sulfate, and 1 mM glutamax®] supplemented with or without 100 mM CLA and with or without 3.03 mM L-carnitine for 22 to 24 h at 38.5°C in a humidified atmosphere of 5% CO2. The proportion of oocytes that cleaved was determined on Day 3 after insemination, and the proportion of oocytes developing to the blastocyst and advanced blastocysts stages (expanded, hatching, and hatched) was assessed on Day 7. Blastocyst and expanded blastocyst stage embryos (n = 270) were harvested on Day 7 and subjected to controlled-rate freezing following equilibration in 1.5 M ethylene glycol. Embryos were thawed and then cultured for 72 h in SOF-BE1 (Fields et al. 2011) supplemented with 10% (vol/vol) fetal bovine serum and 50 μM dithiothreitol. Post-thaw re-expansion and hatching rates were determined at 24, 48, and 72 h. The experiment was replicated 5 times. There was no effect of supplementation of maturation medium with either CLA or L-carnitine on the proportion of oocytes that cleaved at Day 3 or that developed to the blastocyst and advanced blastocyst stages at Day 7 after insemination. There was no interaction between CLA and L-carnitine affecting cleavage rate or embryo development. Supplementation of maturation medium with L-carnitine did not affect post-thaw re-expansion or hatching rates. In contrast, treatment with CLA during maturation reduced (P < 0.05) post-thaw re-expansion (24 h: 75.2 ± 3.8% v. 60.3 ± 4.1%; 48 h: 82.0 ± 3.4% v. 64.9 ± 4.0%; 72 h: 78.9 ± 3.6% v. 65.9 ± 4.0%, respectively) and hatching (24 h: 33.7 ± 4.2% v. 23.5 ± 3.6%; 48 h: 61.1 ± 4.3% v. 44.0 ± 4.2%; 72 h: 62.6 ± 4.3% v. 50.2 ± 4.2%, respectively) rates at all time points. There was no interaction between CLA and L-carnitine affecting post-thaw viability. In conclusion, supplementation of maturation medium with L-carnitine did not affect embryo development or post-thaw viability. Although addition of CLA during maturation did not affect embryo development, post-thaw cryotolerance was reduced following CLA supplementation. There was no beneficial effect of supplementing maturation medium with both CLA and L-carnitine on embryo development or post-thaw cryosurvival.

Time course of the meiotic arrest in sheep cumulus–oocyte complexes treated with roscovitine

Zygote ◽  
2015 ◽  
Vol 24 (2) ◽  
pp. 310-318 ◽  
Author(s):  
Letícia Ferrari Crocomo ◽  
Wolff Camargo Marques Filho ◽  
Camila Louise Ackermann ◽  
Daniela Martins Paschoal ◽  
Midyan Daroz Guastali ◽  
...  
Keyword(s):  
Exposure Time ◽  
Cell Expansion ◽  
Time Course ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Nuclear Maturation ◽  
Maturation Medium

SummaryTemporary meiosis arrest with cyclin-dependent kinases inhibitors has been proposed in order to improve the quality of in vitro matured oocytes. In sheep, however, this phenomenon has been rarely investigated. Therefore, the present study aimed to evaluate the effect of different incubation times with roscovitine on nuclear maturation and cumulus cell expansion of sheep cumulus–oocyte complexes (COCs). For this, COCs were cultured for 0, 6, 12 or 20 h in basic maturation medium (Control) containing 75 μM roscovitine (Rosco). After, they were in vitro matured (IVM) for 18 h in the presence of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). At the end of each treatment, cumulus cell expansion and nuclear maturation were assessed under a stereomicroscope and by Hoechst 33342 staining, respectively. In the Control and Rosco groups, the absence of cumulus cell expansion prevailed at 0, 6, 12 and 20 h. After IVM for 18 h, total cumulus cell expansion in the Rosco treatments was dependent on the exposure time to roscovitine. A significantly high percentage of oocytes treated with roscovitine for 6 h (87%), 12 h or 20 h (65%) were arrested at the germinal vesicle (GV) stage. In contrast, 23% GVBD, 54% metaphase I (MI) and 61% MII oocytes were observed in the Control groups at 6, 12 and 20 h, respectively. In all treatments, a significant percentage of oocytes reached MII after IVM for 18 h. Therefore, roscovitine reversibly arrested the meiosis of sheep oocytes during different culture times with the maximal efficiency of meiotic inhibition reached at 6 h. In addition, reversibility of its inhibitory action on cumulus cells was exposure-time dependent.

scholarly journals Identifying Biomarkers of Autophagy and Apoptosis in Transfected Nuclear Donor Cells and Transgenic Cloned Pig Embryos

2019 ◽  
Vol 19 (1) ◽  
pp. 127-146
Author(s):  
Ju-Young Lee ◽  
Sang Hwan Kim ◽  
Jong Taek Yoon
Keyword(s):  
Embryo Development ◽  
Blastocyst Stage ◽  
Developmental Potential ◽  
Transfected Cells ◽  
Autophagy Inhibitor ◽  
Dermal Cell ◽  
Donor Cells ◽  
Significant Difference ◽  
Dermal Cells

AbstractIn this study, we first investigated the effects of 3-methyladenine (3-MA), an autophagy inhibitor, and the inducer – rapamycin (RAPA) on the incidence of programmed cell death (PCD) symptoms during in vitro development of porcine somatic cell nuclear transfer (SCNT)-derived embryos. The expression of autophagy inhibitor mTOR protein was decreased in porcine SCNT blastocysts treated with 3MA. The abundance of the autophagy marker LC3 increased in blastocysts following RAPA treatment. Exposure of porcine SCNT-derived embryos to 3-MA suppressed their developmental abilities to reach the blastocyst stage. No significant difference in the expression pattern of PCD-related proteins was found between non-transfected dermal cell and transfected dermal cell groups. Additionally, the pattern of PCD in SCNT-derived blastocysts generated using SC and TSC was not significantly different, and in terms of porcine SCNT-derived embryo development rates and total blastocyst cell numbers, there was no significant difference between non-transfected cells and transfected cells. In conclusion, regulation of autophagy affected the development of porcine SCNT embryos. Regardless of the type of nuclear donor cells (transfected or non-transfected dermal cells) used for SCNT, there was no difference in the developmental potential and quantitative profiles of autophagy/apoptosis biomarkers between porcine transgenic and non-transgenic cloned embryos. These results led us to conclude that PCD is important for controlling porcine SCNT-derived embryo development, and that transfected dermal cells can be utilized as a source of nuclear donors for the production of transgenic cloned progeny in pigs.

140 EFFECT OF A NOVEL BOVINE EMBRYO CULTURE MEDIUM TO IMPROVE BLASTOCYST DEVELOPMENT

2013 ◽  
Vol 25 (1) ◽  
pp. 217
Author(s):  
R. F. Gonçalves ◽  
C. Figueiredo ◽  
M. A. Achilles
Keyword(s):  
Culture Medium ◽  
Apoptotic Cell ◽  
Cell Number ◽  
Total Cell ◽  
Cell Numbers ◽  
Group 2 ◽  
Group 1 ◽  
Hatching Rates

There are still immense differences in the quality of in vitro-produced embryos compared to their in vivo-generated counterparts. These differences include a higher sensitivity of in vitro-produced embryos towards cryopreservation. The quality of such embryos has been evaluated using various parameters like morphological examination, assessment of total cell numbers, or pregnancy rates after transfer. In the present study, the effects of glycine, alanine, taurine, and glutamine addition to SOF (Achilles Genetics culture medium, Achilles Genetics®, Garça, SP, Brazil) on the in vitro development (cleavage and blastocyst rates) and quality (total cell and apoptotic cell numbers) of bovine embryos were determined. Ovaries of Nelore cows were obtained from a slaughterhouse. Cumulus–oocyte complexes (COC) were collected from follicles ≥4 mm in diameter, matured in TCM-199, and fertilized with frozen–thawed Nelore bull semen (IVF = Day 0). On Day 1, presumptive zygotes were cultured in SOF supplemented with fetal bovine serum (FBS, group 1, n = 550) or in Achilles Genetics culture medium (SOF supplemented with Achilles Mixture and FBS, group 2, n = 557) at 38.5°C and 5% CO2 in air until Day 9. Embryos were evaluated during culture: at Day 3 cleavage rates, at Day 7 blastocyst rates, and on Day 9 hatching rates. Experiments were replicated 5 times, analysed using ANOVA, followed by a comparison of means by Tukey test (P ≤ 0.05). Blastocysts at Day 8 from Group 1 (n = 75) and Group 2 (n = 75) were fixed and permeabilized for TUNEL assay (DeadEndTM Florimetric TUNEL System, Promega, Madison, WI, USA), according to the manufacturer instructions. Total cell number, apoptotic cell number, and apoptotic cell index (calculated by dividing the apoptotic cell number by total cell number) were analyzed by analysis of variance and means were compared by Student Newman Keuls test. The threshold of significance was set at P ≤ 0.05. Cleavage rates were 79.2 ± 2.5 for group 1 and 91.0 ± 2.5 for group 2. Blastocyst and hatching rates (calculated on the total of zygotes) for group 2 (47.4 ± 2.8; 82.1 ± 1.5) were significantly greater than for group 1 (39.8 ± 2.8; 74.3 ± 1.5). The total cell numbers were not different (P > 0.05) between group 1 (112.7 ± 2.9) and group 2 (111.1 ± 2.7). Blastocysts from group 2 showed lower (P < 0.05) number of apoptotic cells (10.7 ± 1.2) than those from group 1 (20.9 ± 1.2). These results indicate that the addition of glycine, alanine, taurine, and glutamine to SOF (Achilles Mixture) may be an important energy source for the bovine blastocyst and could act synergistically to enhance embryo development to the hatching stage and embryo quality. Financial support from CNPq and FAPESP.

26 EFFECT OF TREATMENT OF BOVINE DONOR CELLS WITH MOUSE EMBRYONIC STEM CELL EXTRACT ON THE DEVELOPMENT OF EMBRYOS AFTER NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 119
Author(s):  
S. Akagi ◽  
E. Mizutani ◽  
Y. Inaba ◽  
M. Kaneda ◽  
T. Somfai ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Control Group ◽  
In Vitro Development ◽  
Donor Cells ◽  
Effect Of Treatment ◽  
Vitro Development

The efficiency of somatic cell cloning is very low, probably because of incomplete reprogramming of the somatic cell nucleus. In recent studies, it is suggested that transient exposure of donor somatic cells to mouse embryonic stem cell (ESC) extract enhances pluripotency of the cells in vitro (Bru et al. 2008 Exp. Cell Res. 314, 1634–1642; Xu et al. 2009 Anat. Rec. 292, 1229–1234). In the present study, we examined the effect of treatment of donor cells with mouse ESC extract on the in vitro development of bovine NT embryos. First, in order to examine effect of treatment of donor cells with streptolysin O (SLO), which reversibly permeabilizes the plasma membrane, we compared the in vitro development of NT embryos using donor cells treated with 5 μg mL–1 SLO (SLO group) and untreated donor cells (control group). As donor cells for NT, bovine fibroblast cells of passages 3 to 5 were used. Fibroblasts were treated with 5 μg mL–1 SLO for 45 min, and then incubated for resealing in DMEM including 2 mM CaCl2 for 60 min. NT was performed as previously described (Akagi et al. 2003 Mol. Reprod. Dev. 66, 264–272). After in vitro culture for 8 days, blastocyst formation and cell number of blastocysts were examined. There were no significant differences between SLO and control groups in the fusion rate (80% and 72%, respectively), cleavage rate (60% and 65%, respectively), developmental rate to the blastocyst stage of NT embryos (31% and 28%, respectively), and blastocyst cell number (127 ± 6 and 112 ± 14, respectively). These results suggest that SLO treatment of donor cells has no negative effect on the in vitro development of NT embryos. Next, we examined the in vitro developmental ability of NT embryos using donor cells treated with mouse ESC extract (ES extract group). After SLO treatment for 45 min, permeabilized fibroblast cells were treated with mouse ESC extract for 45 min, and then incubated in DMEM including 2 mM CaCl2 for 60 min, and used for producing NT embryos. There were no differences between ES extract and control groups in the fusion rate (68% and 69%, respectively), cleavage rate (86.7% and 80.6%, respectively), and developmental rate to the blastocyst stage of NT embryos (39.8% and 43.5%, respectively). The cell number of NT embryos at the blastocyst stage in ES extract group (201 ± 30) was significantly (t-test; P < 0.05) higher than that in control group (140 ± 14). In conclusion, treatment of bovine donor cell with mouse ESC extract did not affect the in vitro developmental ability of NT embryos, but improved the quality of blastocysts.

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