31 DEVELOPMENT AND APOPTOSIS IN BOVINE CLONED EMBRYOS RECONSTRUCTED WITH OOCYTES COLLECTED BY REPEATED OVUM PICKUP SESSIONS OR FROM SLAUGHTERED COW OVARIES

2011 ◽  
Vol 23 (1) ◽  
pp. 122
Author(s):  
L. S. A. Camargo ◽  
M. M. Pereira ◽  
C. C. R. Quintao ◽  
J. N. S. Sales ◽  
L. T. Iguma ◽  
...  
Keyword(s):  
Apoptotic Cell ◽  
Bos Indicus ◽  
Cell Number ◽  
Total Cell ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Cell Index ◽  
Sh Groups ◽  
Sh Group

The oocyte has important components for nuclear reprogramming and its cytoplasmic background may influence the somatic cell nuclear transfer success. The current study attempted to evaluate the competence of cytoplasm from oocytes recovered by repeated ovum pickup (OPU) in living cows (OPU group) or obtained from ovaries collected at slaughterhouse from unknown source crossbred cows (SH group) to produce nuclear-transferred bovine embryos. For the OPU group, oocytes were recovered from 4 Bos indicus × Bos taurus crossbred cows in 4 repeated OPU sessions. Oocytes of OPU and SH groups were matured in vitro for 17 to 18 h, denuded and exposed to Hoechst 33342 (Sigma, St. Louis, MO, USA) and cytochalasin (Sigma) before enucleation. Embryos of OPU (n = 100) and SH (n = 105) groups were reconstructed with somatic cells from adult Gyr (Bos indicus) cow, fused with double electric pulse of 2.4 kV cm–1 for 30 μs and activated with ionomycin (Sigma) and 6-DMAP (Sigma). Embryos were cultured in CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell, Campinas, Brazil) under 5% CO2, 5% O2, and 90% N2 at 38.5°C. Cleavage and blastocyst rates were evaluated at 72 h and 168 h post-activation, respectively. Blastocysts at 168 h post-activation were fixed and permeabilized for TUNEL assay (DeadEnd™ Fluorimetric TUNEL System, Promega, Madison, WI, USA), according to the manufacturer instructions. IVF bovine blastocysts (IVF group; n = 245) obtained with oocytes of slaughtered cows were used as control group. Fusion, cleavage, and blastocyst rates were analysed by chi-square test and total cell number, apoptotic cell number, and apoptotic cell index (calculated by dividing the apoptotic cell number by total cell number) were analysed by ANOVA. There were no differences (P > 0.05) in fusion (71.0% and 61.0%), cleavage (74.6% and 78.1%) or blastocyst (32.3% and 31.2%) rates between OPU and SH groups, respectively, but both groups presented greater (P < 0.05) blastocyst rates than the IVF group (15.1%). Total cell number (80.66 ± 5.36 and 82.10 ± 4.79), apoptotic cell number (12.66 ± 3.20 and 15.60 ± 3.04), and apoptotic cell index (0.15 ± 0.03 and 0.20 ± 0.04) were also similar (P > 0.05) between OPU and SH groups, respectively. However, apoptotic cell number (7.40 ± 0.93) and apoptotic cell index (0.07 ± 0.01) were lower (P < 0.05) in the IVF group than the SH group and similar (P > 0.05) to the OPU group. In conclusion, oocytes cytoplasm from both groups (OPU and SH) have the same potential to produce nuclear-transferred bovine embryos but only blastocysts from the OPU group present apoptosis levels similar to its in vitro-fertilized counterpart. Financial support: Fapemig and CNPq.

147 THE DEVELOPMENTAL CHARACTERISTICS OF IN VITRO-PRODUCED CATTLE-WISENT (BOS TAURUS-BISON BONASUS) HYBRID EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 182
Author(s):  
E. N. Shedova ◽  
G. N. Singina ◽  
V. A. Bagirov ◽  
N. A. Zinovieva
Keyword(s):  
Bos Taurus ◽  
Apoptotic Cell ◽  
Cell Number ◽  
Total Cell ◽  
European Bison ◽  
Bison Bonasus ◽  
Epididymal Sperm ◽  
Total Cell Number ◽  
Cell Ratio

Interspecies hybrids are important resources for research and agriculture. Therefore, the aim of this study was to evaluate development, quality, and viability of embryos produced in vitro using cattle (Bos taurus) oocytes and European bison (Bison bonasus) epididymal sperm. The epididymes were obtained following a forced slaughter of one bull aged 7 years. The sperm was collected by scraping the inner surface of the epididymes, diluted with the cryopreservation medium, and equilibrated for 4 h at 4°C. Thereafter, sperm aliquots (0.2 mL) were frozen in liquid nitrogen vapor for 5 min and then plunged into liquid nitrogen for storage. Prior to fertilization, frozen semen was thawed in pre-warmed medium for 1 min at 37°C and prepared by the swim-up method. The frozen-thawed ejaculated sperm from the Russian Black Pied bulls was used as a positive control. Slaughterhouse-derived cumulus-oocyte complexes were matured for 24 h in TCM-199 supplemented with 10% FCS, 0.2 mM sodium pyruvate, 10 μg mL−1 porcine FSH, and 10 μg mL−1 ovine LH. Matured oocytes (35–40 oocytes per group) were co-incubated for 18 h with homologous (n = 266 oocytes) or heterologous (n = 292 oocytes) sperm (spermatozoa/mL) in 500 µL of TALP containing 10 μg mL−1 heparin, 20 μM penicillamine, 10 μM hypotaurine, 1 μM epinephrine, and 0.1% minimal essential medium nonessential amino acids. After IVF, the oocytes were cultured in CR1aa medium (Rosenkrans 1994 J. Anim. Sci. 72, 434–437) to the blastocyst stage. All the cultures were performed at 38.5°C and 5% CO2 in humidified air. At Days 2 and 7 after insemination, the cleavage and blastocyst rates were determined. In addition, a part of obtained blastocysts was fixed with 4% paraformaldehyde, and the total cell number and apoptotic cell ratio were determined by 4’,6-diamidino-2-phenylindole and TUNEL staining. The remaining blastocysts were cultured up to Day 10, and the hatching rates were assessed. The data (3–5 replicates) were analysed by ANOVA. The cleavage rates did not differ among both male species (72.4 and 77.1%). Furthermore, no significant effects of interspecies fertilization on the blastocyst rate or total cell number per blastocyst were found (27.4 ± 1.6% and 77.0 ± 5.7 for cattle embryos and 26.2 ± 1.9% and 83.1 ± 8.9 for cattle-wisent hybrid embryos). On the other hand, the significant differences between homologous and heterologous fertilization were detected in the rate of hatched blastocysts (60.3 ± 5.1 v. 38 ± 2.9, P < 0.05) and apoptotic cell ratio 7.3 ± 0.8 v. 11.6 ± 1.04, P < 0.05). Our findings demonstrate that hybrid embryos produced by IVF of bovine oocytes with the epididymal sperm of European bison can be developed up to advanced blastocyst stages. However, the hybrid embryos have a lower quality and viability than cattle embryos. Research was supported by the Program of Presidium of the Russian Academy of Science, project no. IV.13.3.

scholarly journals Effect of cysteamine supplementation during in vitro culture of early stage bovine embryos on blastocyst rate and quality

2012 ◽  
Vol 81 (3) ◽  
pp. 229-234 ◽  
Author(s):  
Martina Lojkic ◽  
Iva Getz ◽  
Marko Samardžija ◽  
Mario Matkovic ◽  
Goran Bacic ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Oviductal Fluid

The aim of this study was to evaluate whether the addition of cysteamine to the in vitro culture media enhances the yield, hatching rate, total cell number and inner cell mass/total cell number ratio of bovine embryos. A total of 933 bovine oocytes collected from ovaries of 60 slaughtered donors were subjected to in vitro maturation and in vitro fertilization. Following fertilization, embryos were cultured in synthetic oviductal fluid without glucose. After 24 h embryos were transferred into synthetic oviductal fluid with 1.5 mM glucose and 0 (control), 50, 100 and 200 µM of cysteamine. After 48 h, the embryos were transferred into synthetic oviductal fluid with glucose but without cysteamine and cultured until Day 9. The number of cleaved embryos on Day 2, the total number of blastocysts on Day 7 and the number of hatched blastocysts on Day 9 were calculated. Differential staining of inner cell mass and trophectoderm cells of blastocysts were performed on Day 7 and Day 9 of in vitro culture. Supplementation of in vitro culture media with 100 µM cysteamine increased the blastocyst yield (P < 0.05) without affecting the hatching rate. Furthermore, the embryos cultured in the presence of 100 µM cysteamine had significantly higher number of inner cell mass cells (P < 0.05) and the proportion of inner cell mass cells (P < 0.05) compared with the controls. The results of the present study demonstrated that the addition of 100 µM cysteamine to the in vitro culture media improved blastocyst production rate and enhance embryo quality, which could lead to the improvement of the in vitro culture system for bovine embryos.

scholarly journals 95 EFFECT OF ESTROUS COW SERUM ON SURVIVAL OF IN VITRO-PRODUCED BOVINE EMBRYOS AFTER SLOW FREEZING OR VITRIFICATION

2005 ◽  
Vol 17 (2) ◽  
pp. 198
Author(s):  
N. Mucci ◽  
J. Aller ◽  
P. Ross ◽  
G. Kaiser ◽  
J. Cabodevila ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Cell Number ◽  
Total Cell ◽  
T Test ◽  
Slow Freezing ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Embryo Survival

Until now, the major obstacle associated with the extensive use of in vitro-produced bovine embryos is the lack of suitable methods to cryopreserve them. At least two approaches exist for overcoming this problem. One is to adjust cryopreservation methods to the requirements of these embryos, and the other is to improve embryo quality by using an appropriate in vitro environment for embryo production. The objective of this study was to determine the effect of estrous cow serum (ECS) during in vitro culture on embryo survival after cryopreservation by slow freezing or vitrification. Cumulus-oocytes complexes were in vitro-matured and fertilized as previously described (Ferre et al. 2003 Theriogenology 59, 301 abst). Presumptive zygotes were denuded from cumulus cells and cultured in groups of 50 in 400 μL drops of CR1aa medium. Seventy-two hour post-insemination (PI) embryos were randomly separated into three groups. Each group was then cultured in CR1aa + 5% ECS (without BSA; CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA), or CR1aa + 5% ECS + 3 mg/mL BSA (CR1-ECS-BSA). Embryos were cultured under 38.5°C, 5% CO2, 5% O2, and 90% N2. At 7.5 days PI, blastocysts from each group were double stained using propidium iodide and bisbenzimide (Hoechst 33342) to determine damaged cells and total cell number. The remaining embryos were randomly cryopreserved by freezing (1.5 M ethylene glycol; cooled at 0.5°C/min to −35°C) or vitrification (open pulled straw, Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58). After thawing or warming, embryos were cultured in CR1-ECS-BSA to evaluate embryo survival (hatching rate). Data were analyzed by χ2, ANOVA and Student's t-test (SAS Institute, Inc., Cary, NC, USA). Total cell number was higher in embryos cultured in CR1-ECS than in CR1-BSA or CR1-ECS-BSA (CR1-ECS: 142.1 ± 4.7, n = 23 vs. CR1-BSA 124.7 ± 4.9, n = 21, and CR1-ECS-BSA 125.8 ± 4.5, n = 25; t-test, P < 0.05). No differences were found in percent of damaged cells (CR1-ECS: 0.7%; CR1-BSA: 1.8%; CR1-ECS-BSA: 0.7%). Blastocyst survival after thawing was affected by cryopreservation methods and culture media (P < 0.01, Table 1). No interaction was found between both factors. In conclusion, under our experimental conditions elimination of ECS from CR1aa medium improves embryo cryotolerance. Vitrification allows for higher survival rates, regardless of the presence of serum during embryo culture. Table 1. Effect of cryopreservation method and serum supplementation during embryo culture on survival rate of in vitro-produced bovine embryos

scholarly journals 158APOPTOSIS IN IN VITRO PRODUCED BOVINE EMBRYOS ACCORDING TO DEVELOPMENTAL KINETICS

2004 ◽  
Vol 16 (2) ◽  
pp. 201 ◽  
Author(s):  
F.V. Meirelles ◽  
K.L. Schwarz ◽  
G.K.F. Merighe ◽  
S.F. Carambula ◽  
Y.F. Watanabe
Keyword(s):  
Rapid Development ◽  
Rna Extraction ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Cell Stage ◽  
Slow Development

Apoptosis has been previously reported in embryos during late pre-implantation development. Fast-developing embryos are known to present higher developmental competence. The aim of the present work was to evaluate the quality of in vitro-produced bovine embryos with fast (8-cells at 48 hours post-insemination (hpi) and slow (8-cells at 90hpi) cleavage and study the correlation of this phenotype with programmed cell death occurrences. Embryos were produced from immature oocytes obtained from slaughtered cow ovaries, after maturation and fertilization, presumed zygotes were cultured in CR2 medium with 10% FCS, together with granulosa cells under 5% CO2 atmosphere. The number of nuclei in the inner cell mass and trophectoderm (ICM/TE), as well as the number of nuclei with fragmented DNA, were estimated by applying differential staining and TUNEL, respectively; data were analyzed by ANOVA (JMP—SAS Institute). To test the expression of apoptosis regulating genes, a pool of fifty 8-cell embryos from each group (fast and slow) were collected. After RNA extraction and reverse transcriptase reaction, cDNA was amplified with Bax and Bcl2 primers, individually. Results indicated, as expected, higher quality in fast-cleaving embryos, estimated by the number of ICM nuclei (20.8±1.4 and 15.6±2.1—P≤0.05); however, the number of TE didn’t show significant differences (54.9±2.4 and 53.2±3.8); the same was observed for total cell number (75.7±2.8 and 68.8±4.4). The frequency of blastocyst TUNEL-positive nuclei as an estimate of total cell number was significantly larger in the slow group when compared to the rapid development group (19.0±2.5% and 8.5±1.4%, respectively, P≤0.05). The greater proportion of morphologic abnormal nuclei in both groups was located in the ICM, and may explain the lower number of ICM nuclei in slow developing embryos. Hence, embryos of slow development show TUNEL-positive blastomeres at the 8-cell stage, but no fragmented nuclei were observed in embryos at 48hpi. Bax and Bcl2 cDNA amplification showed that both mRNAs were constitutively present at the 8-cell stage in both groups. It can be concluded that in vitro-produced bovine blastocysts, with slow development to the 8-cell stage, present lower quality compared with fast development homologues, estimated by mean number of ICM nuclei, as well as nuclei fragmentation in blastomeres (TUNEL-positive). There is a difference in fragmented nuclei proportion between both groups at the 8-cell stage, but this result may be biased by the numbers of hours in culture. It was possible to demonstrate the presence of mRNA for pro (Bax) and anti-apoptotic (Bcl2) genes in slow- and fast-developing embryos at the 8-cell stage, and the future determination of the ratio between these two transcripts may allow the evaluation of the participation of pre-transcriptional regulation of these genes on the induction of DNA fragmentation. Financial support: Grant 99/12351-3 FAPESP São Paulo, Brazil.

84 THE EFFECT OF β-MERCAPTOETHANOL ON CLEAVAGE RATES, DEVELOPMENTAL COMPETENCE AND QUALITY OF IN VITRO PRODUCED BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 156 ◽  
Author(s):  
E. R. Lliteras ◽  
M. Chong ◽  
S. Andries ◽  
E. Merckx ◽  
E. P. A. Jorssen ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture Media ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Dapi Staining ◽  
Total Cell Number

The production of excessive levels of reactive oxygen species can be a major problem during in vitro embryo culture. Although studies have shown that supplementation with exogenous antioxidants can improve embryo quality, the results are controversial among researchers. In this study, we examined the effects of different concentrations of β-mercaptoethanol (β-ME) added to the culture media, on cleavage rates, the quality and developmental competence of in vitro-produced bovine embryos. The embryos were produced in vitro as described previously (Van Hoeck et al., 2013). Briefly, in total, 753 grade I cumulus–oocyte complexes (COC) from 2- to 6-mm-diameter follicles were matured in groups of 50 in 500 μL of TCM with 20 ng mL–1 EGF for 24 h, fertilized in groups of 100 in 500 μL of fertilization medium for 20 h (5% CO2, 38.5°C). Presumptive zygotes were denuded and randomly assigned to 4 treatments with different concentrations of β-ME: 0 μM (control), 50 μM, 100 μM, and 150 μM. They were cultured in groups of  ±25 in 50 μL of SOF supplemented with ITS (10 μg mL–1 insulin; 5.5 μg mL–1 transferrin; 6.7 ng mL–1 selenium) and 2% BSA and covered with mineral oil (5% O2, 5% CO2, 38.5°C). At 48 h post-insemination (p.i.), cleavage rate was evaluated and expressed as the number of cleaved embryos on total number of oocytes. At Day 7 p.i., blastocyst rate was determined (number of blastocysts on total number of oocytes), blastocysts were fixed in 4% paraformaldehyde, and total cell number was determined by DAPI staining. Data were analysed by ANOVA and post hoc test. Comparable cleavage rates were obtained in treated groups: control (80.8%), 50 μM (77.7%), 100 μM (77.9%), and 150 μM (73.6%; P > 0.05). Also, no significant effect of treatment could be found on blastocyst rates: control (36%), 50 μM (36.5%), 100 μM (38.4%), and 150 μM (30.4%). The total cell number per blastocyst increased significantly (P < 0.05) using 100 μM of β-ME compared with the controls (158.0 ± 24.3 v. 123.2 ± 9.72, respectively). These results suggest that the inclusion of 100 μM β-ME during in vitro embryo culture could be used for production of high quality bovine blastocysts.

85 THE EFFECT OF 17α-ETHINYL ESTRADIOL EXPOSURE OF IN VITRO-CULTURED BOVINE MORULAE ON SUBSEQUENT EMBRYONIC DEVELOPMENT AND QUALITY

2014 ◽  
Vol 26 (1) ◽  
pp. 156
Author(s):  
E. P. A. Jorssen ◽  
L. Jordaens ◽  
E. Merckx ◽  
S. Andries ◽  
J. L. M. R. Leroy ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Apoptotic Cell ◽  
Cell Number ◽  
Total Cell ◽  
Total Cell Number ◽  
Cell Ratio ◽  
Treatment Groups ◽  
Morula Stage ◽  
Solvent Control

Research has shown that 17-α-ethinyl oestradiol (EE2), an important component of most oral birth-control pills, acts as a xeno-oestrogen after being released into the environment through urine and feces. Although this emphasizes the need for the evaluation of its toxicity, several oocyte and embryo-toxic effects have been reported (Beker-Van Woudenberg et al. 2012). Therefore, the aim of the present study was to evaluate the effect of EE2 exposure during bovine early embryonic development, specifically at the morula stage (18 h), on subsequent embryonic development and quality. Bovine cumulus–oocyte complexes (COC) from 2- to 6-mm-diameter follicles were matured in groups of 50 in 500 μL of TCM with 20 ng mL–1 epidermal growth factor (EGF) for 24 h and subsequently fertilized in groups of 100 in 500 μL of fertilization medium for 22h (5% CO2, 38.5°C). Presumptive zygotes were denuded and cultured in groups of  ±25 in 50 μL of SOF with ITS (5 μg mL–1 insulin, 5 μg mL–1 transferrin, 5 ng mL–1 selenium) and 2% BSA, covered with mineral oil (5% O2, 5% CO2, 38.5°C). Subsequently, embryonic developmental stage was determined at 135 h post-insemination (p.i.). Sole embryos at morula stage were selected and randomly allocated to treatment groups (n) divided over 5 replicates: (1) Control (67), (2) solvent control: 0.1% ethanol (49), (3) 10 ng mL–1 EE2 (49), or (4) 10 μg mL–1 EE2 (63). The morulas were cultured individually in 30 μL of standard SOF medium supplemented with the desired concentrations ethanol or EE2 (5% O2, 5% CO2, 38.5°C) in 96-well half-area culture plates, without oil coverage for 18 h. Following exposure, embryos were cultured singly in standard culture medium for 2 more days. Subsequently, developmental competence was evaluated and blastocyst rates calculated (blastocyst rate = total blastocyst/number of grade 1 selected morula). Expanded (EB) and hatched blastocysts (HB) were fixed with 4% paraformaldehyde, and total cell number and apoptotic cell ratio were determined by DAPI and TUNEL staining (13 EB and 7 HB per treatment). Comparable blastocyst rates were obtained in all treatment groups: solvent control (77.8%), 10 ng mL–1 EE2 (69.0%), and 10 μg mL–1 EE2 (84.0%) compared with the controls (88.2%; P > 0.05; binary logistic regression). In addition, no significant effect of treatment could be found on total cell number or apoptotic cell ratio: solvent control (149.70 ± 23.47 and 3.46 ± 1.73), 10 ng mL–1 EE2 (154.75 ± 23.26 and 3.22 ± 1.35), and 10 μg mL–1 EE2 (150.50 ± 26.69 and 4.23 ± 1.85) compared with the controls (145.02 ± 24.71 and 3.07 ± 2.03; P > 0.05; two-way ANOVA). Although our results show no immediate statistical significant effect of short-term EE2 exposure during the morula stage in in vitro culture on subsequent blastocyst development and quality, additional research is necessary to find out if EE2 may affect gene-expression patterns, eventually resulting in still unknown embryotoxic effects that might turn up during later embryonic development.

148 EFFECTS OF Wnt3A SUPPLEMENTATION ON BOVINE BLASTOCYST CELL NUMBER AND ALLOCATION

2010 ◽  
Vol 22 (1) ◽  
pp. 232
Author(s):  
M. D. Goissis ◽  
P. J. Ross ◽  
J. B. Cibelli
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Cell Counts ◽  
Esc Derivation ◽  
Hatching Rates

Derivation of true bovine embryonic stem cells (ESC), as defined by their capacity to form robust teratomas and/or contribute to the germ line in chimeras, has not been achieved despite several attempts. It is possible that failures to derive bonafide bovine ESC are due to the inability of bovine embryonic cells to adapt to in vitro culture conditions that favor ESC derivation. Wnt pathways are involved in pluripotency and self-renewal of mouse and human ESC. Wnt signaling is also required for implantation competence in mouse blastocysts. Given the shared developmental potential between inner cell mass (ICM) and ESC, we hypothesized that Wnt could act on the ICM of bovine embryos increasing its proliferation potential. The objective of this study was to evaluate the effect of post-embryonic genome activation Wnt3A supplementation on blastocyst formation and cell allocation to ICM and trophectoderm (TE). In vitro fertilized bovine embryos at Day 4 of culture in KSOM medium were divided into 3 treatments: Control, no co-culture; co-culture with regular mouse embryonic fibroblasts (MEF); and co-culture with mouse L fibroblasts overexpressing Wnt3A protein (L-Wnt3A, Willert et al. 2003 Nature 423, 448-452). Embryos were cultured until Day 8 when blastocyst and hatching rates were recorded. Then, embryos were submitted to differential staining of ICM and TE by brief exposure to 0.25% Triton X-100 in PBS and staining with bisbenzimide and propidium iodide. Six IVF replications were performed and a total of 39 embryos were counted: 11 for Control, 16 for MEF, and 12 for L-Wnt3A. Only intact embryos after processing were used for cell count. Statistical analysis was performed by ANOVA using PROC MIXED of SAS software (SAS Institute Inc., Cary, NC, USA) in which each IVF was considered as a block with Tukey’s adjustment for mean comparison of rates and Bonferroni adjustments for mean comparison of cell counts. Results for blastocyst rate, hatching rate, ICM, TE, and total cell number are presented in the table below. Different superscript letters within columns indicate significant statistical difference (P < 0.05). These results indicate that L-Wnt3A fibroblast co-culture exerts a positive effect on bovine embryo cell number, resulting in a larger number of ICM cells in bovine embryos, which could be beneficial for ESC derivation attempts. Table 1.Blastocyst and hatching rates, ICM, TE, and total cell number results

scholarly journals Effect of insulin–transferrin–selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation on in vitro bovine embryo development

Zygote ◽  
2016 ◽  
Vol 24 (6) ◽  
pp. 890-899 ◽  
Author(s):  
A.L.S. Guimarães ◽  
S.A. Pereira ◽  
M. N. Diógenes ◽  
M.A.N. Dode
Keyword(s):  
Ascorbic Acid ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Bovine Embryo ◽  
Total Cell Number ◽  
Embryo Production ◽  
Embryo Size ◽  
Blastocyst Rate

SummaryThe aim of this study was to evaluate the effect of adding a combination of insulin, transferrin and selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation (IVM) and in vitro culture (IVC) on in vitro embryo production. To verify the effect of the supplements, cleavage and blastocyst rates, embryo size and total cell number were performed. Embryonic development data, embryo size categorization and kinetics of maturation were analyzed by chi-squared test, while the total cell number was analyzed by a Kruskal–Wallis test (P < 0.05). When ITS was present during IVM, IVC or the entire culture, all treatments had a cleavage and blastocyst rates and embryo quality, similar to those of the control group (P < 0.05). Supplementation of IVM medium with ITS and AA for 12 h or 24 h showed that the last 12 h increased embryo production (51.6%; n = 220) on D7 compared with the control (39.5%; n = 213). However, no improvement was observed in blastocyst rate when less competent oocytes, obtained from 1–3 mm follicles, were exposed to ITS + AA for the last 12 h of IVM, with a blastocyst rate of 14.9% (n = 47) compared with 61.0% (n = 141) in the control group. The results suggest that the addition of ITS alone did not affect embryo production; however, when combined with AA in the last 12 h of maturation, there was improvement in the quantity and quality of embryos produced. Furthermore, the use of ITS and AA during IVM did not improve the competence of oocytes obtained from small follicles.

The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

Development ◽  
1988 ◽  
Vol 102 (4) ◽  
pp. 793-803 ◽  
Author(s):  
V.E. Papaioannou ◽  
K.M. Ebert
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Staining Technique ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Morphological Development ◽  
Total Cell Number

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12–16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched. Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.

Effect of vitrification technique and assisted hatching on rabbit embryo developmental rate

Zygote ◽  
2009 ◽  
Vol 17 (1) ◽  
pp. 57-61 ◽  
Author(s):  
M. Popelková ◽  
Z. Turanová ◽  
L. Koprdová ◽  
A. Ostró ◽  
S. Toporcerová ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Assisted Hatching ◽  
Hatching Rate ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Significant Difference ◽  
Rabbit Embryos

SummaryThe aim of the study was to determine the efficiency of two vitrification techniques followed by two assisted hatching (AH) techniques based on post-thaw developmental capacity of precompacted rabbit embryos and their ability to leave the zona pellucida (hatching) during in vitro culture. The total cell number and embryo diameter as additional markers of embryo quality after warming were evaluated. In vivo fertilized, in vitro cultured 8–12-cell rabbit embryos obtained from superovulated rabbit does were cryopreserved by two-step vitrification method using ethylene glycol (EG) as cryoprotectant or by one-step vitrification method with EG and Ficoll (EG+Ficoll). Thawed embryos were subjected to enzymatic or mechanical AH. Vitrified EG group showed significantly lower (P < 0.05) blastocyst rate (22.5%) and hatching rate (15%) than those vitrified with EG + Ficoll (63 and 63% resp.) and that of control (97 and 97% respectively). Significantly lower values of total cell number (P < 0.05) as well as embryo diameter (P < 0.01) in EG group compared with EG + Ficoll and control group were recorded. No significant difference was found in developmental potential of warmed embryos treated by either mechanical or enzymatic AH. The present study demonstrates that the EG + Ficoll vitrification protocol provides superior embryo survival rates over the EG vitrification protocol for 8–12-cell stage precompacted rabbit embryos. No positive effect of either mechanical or enzymatic AH on the post-thaw viability and quality of rabbit embryos in vitro was observed.

Sign in / Sign up

Export Citation Format

Share Document