scholarly journals QUALITY OF INNER CELL MASS OF EXPANDED BLASTOCYSTS EFFECTS ART OUTCOME MORE THAN TROPHECTODERM QUALITY

2021 ◽  
Vol 116 (3) ◽  
pp. e143
Author(s):  
Sarah Evans ◽  
Paul Knaggs
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Art Outcome

77 Development and quality of in vitro bovine hemi embryos produced by blastomere separation and embryo bisection

2019 ◽  
Vol 31 (1) ◽  
pp. 164
Author(s):  
A. E. Ynsaurralde Rivolta ◽  
M. Suvá ◽  
V. Alberio ◽  
C. Vazquez Echegaray ◽  
A. Guberman ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Monozygotic Twin ◽  
Cell Number ◽  
Control Group ◽  
Cell Stage ◽  
Exact Test ◽  
Development Rates

Bovine monozygotic production of twins became popular in the 1980s as a technique to multiply high value genetics. Moreover, it also became a powerful model for research. Different techniques have been used on bovine embryos obtained by superovulation. In this work, we compared the development rates and quality of monozygotic twin embryos produced by blastomere separation (BS) and embryo bisection (EB) of IVF embryos. To this aim, cumulus-oocytes complexes collected from slaughterhouse ovaries were in vitro matured in TCM 199 containing 10% fetal bovine serum, 10µg mL−1 FSH, 0.3mM sodium pyruvate, 100mM cysteamine, and 2% antibiotic-antimycotic for 24h, at 6.5% CO2 in humidified air and 38.5°C. The IVF was performed with 16×106 spermatozoa per mL for 5h. Afterward, presumptive zygotes were cultured in SOF medium for 7 days at 38.5°C and 5% O2. After 24h of culture, blastomeres of 2-cell stage embryos (N=114) were separated and each one was cultured individually in a microwell for 7 days. Embryo bisection (N=179) was performed manually on Day-7 blastocysts previously depleted of their zonae pellucidae, under stereoscopic microscope. Hemi embryos were cultured for 24h and then twins and single blastocyst rates were calculated. For quality assessment, diameter, total and inner cell mass (ICM) cell number of hemi embryos (BS: 6 couples; ES: 10 couples) and the control group (C: 11) were evaluated. The ICM cell number was measured by immunofluorescence staining using SOX2 antibody and the percentage of ICM and trophectoderm (TE) cells was calculated. The results were analysed using Fisher’s exact test and ANOVA with mean comparison using Tukey’s test (P=0.05). No statistical differences were found in blastocyst rates of twins and single hemi embryos produced by BS (28 and 25%) or EB (23 and 32%). Blastocyst diameter was similar between groups and control. Hemi embryos exhibited lower total and ICM cell number than control (BS: 43±18, EB: 57±14v. C: 93±35 and BS: 16±7, EB: 12±8v. C: 34±19). However, BS hemi embryos had higher ICM and lower TE percentage (40/60%) compared with the EB group (20/80%). The control group did not differ with hemi embryo treatments for ICM and TE (30/70%). Our preliminary results have indicated that although the development rates of hemi embryos produced in vitro were similar between both techniques, blastomere separation generates better quality embryos than blastocyst bisection.

Putrescine supplementation during in vitro maturation of aged mouse oocytes improves the quality of blastocysts

2017 ◽  
Vol 29 (7) ◽  
pp. 1392 ◽  
Author(s):  
Dandan Liu ◽  
Guolong Mo ◽  
Yong Tao ◽  
Hongmei Wang ◽  
X. Johné Liu
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
In Vitro Fertilisation ◽  
Aged Mouse ◽  
Developmental Potential ◽  
The Impact

Mouse ovaries exhibit a peri-ovulatory rise of ornithine decarboxylase and its product putrescine concurrent with oocyte maturation. Older mice exhibit a deficiency of both the enzyme and putrescine. Peri-ovulatory putrescine supplementation in drinking water increases ovarian putrescine levels, reduces embryo resorption and increases live pups in older mice. However, it is unknown if putrescine acts in the ovaries to improve oocyte maturation. This study examined the impact of putrescine supplementation during oocyte in vitro maturation (IVM) on the developmental potential of aged oocytes. Cumulus–oocyte complexes from 9–12-month-old C57BL/6 mice were subjected to IVM with or without 0.5 mM putrescine, followed by in vitro fertilisation and culture to the blastocyst stage. Putrescine supplementation during IVM did not influence the proportion of oocyte maturation, fertilisation or blastocyst formation, but significantly increased blastocyst cell numbers (44.5 ± 1.9, compared with 36.5 ± 1.9 for control; P = 0.003). The putrescine group also had a significantly higher proportion of blastocysts with top-grade morphology (42.9%, compared with 26.1% for control; P = 0.041) and a greater proportion with octamer-binding transcription factor 4 (OCT4)-positive inner cell mass (38.3%, compared with 19.8% for control; P = 0.005). Therefore, putrescine supplementation during IVM improves egg quality of aged mice, providing proof of principle for possible application in human IVM procedures for older infertile women.

Effect of anisomycin, a protein synthesis inhibitor, on thein vitrodevelopmental potential, ploidy and embryo quality of bovine ICSI embryos

Zygote ◽  
2016 ◽  
Vol 24 (5) ◽  
pp. 724-732 ◽  
Author(s):  
María Elena Arias ◽  
Raúl Sánchez ◽  
Ricardo Felmer
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Developmental Rate ◽  
Fertilization Rate ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Protein Synthesis Inhibitor ◽  
Oocyte Activation ◽  
Synthesis Inhibitor

SummaryIncreasing the efficiency of intracytoplasmic sperm injection (ICSI) in domestic animals has been attempted by many researchers, however embryonic development to the blastocyst stage remains low compared with that ofin vitrofertilization (IVF) embryos. One of the main problems observed in cattle is inadequate oocyte activation after ICSI. The present study compared the effect of cycloheximide (CHX), 6-dimethylaminopurine (DMAP), and anisomycin (ANY) on the fertilization rate, development, ploidy and quality of bovine embryos generated by ICSI. Although no differences were observed between treatments in terms of cleavage, higher blastocyst rates were observed for ANY (37.3%) compared with CHX (21.8%,P< 0.05) and DMAP (28.6%,P> 0.05) treatments. No differences were observed in the quality of embryos as assessed by the total number of cells, their distribution to the different embryo compartments [inner cell mass (ICM) and trophectoderm (TE)], the proportion of ICM cells to the total cell numbers and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive cells. Similarly, no differences were observed in the normal ploidy of embryos (56, 67, and 55%) for ANY, CHX and DMAP, respectively. However, higher fertilization rates were observed for ANY (75%) and CHX (87%) treatments compared with DMAP (35%). In conclusion, ANY showed a superior developmental rate compared with CHX treatment. Although no significant differences were observed compared with an improved protocol of DMAP (2Io-DMAP), the lower fertilization rate recorded with DMAP strongly suggests that ANY could be a better alternative for oocyte activation than traditional chemical compounds used currently in ICSI.

High hydrostatic pressure treatment improves the quality of in vitro-produced ovine blastocysts

2011 ◽  
Vol 23 (6) ◽  
pp. 809 ◽  
Author(s):  
Luisa Bogliolo ◽  
Federica Ariu ◽  
Giovanni Leoni ◽  
Stefania Uccheddu ◽  
Daniela Bebbere
Keyword(s):  
Hydrostatic Pressure ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Lower Percentage ◽  
Control Group ◽  
Hatching Rate ◽  
Embryo Survival

Exposure to sub-lethal hydrostatic pressure (HP) treatment is emerging as an approach to improve the general resistance of gametes and embryos to in vitro conditions. The present study was aimed to evaluate the effect of HP treatment on in vitro-produced ovine blastocysts. Experiment 1 was aimed to define optimal treatment parameters: two different HP treatments were applied to blastocysts and embryo survival was evaluated. In Experiment 2, HP parameters (40 MPa, 70 min, 38°C) selected in Experiment 1 were used to treat blastocysts. Embryo quality was assessed and compared with untreated controls by counting total cell number, the inner cell mass (ICM) and trophectoderm (TE) cells and by evaluating nuclear picnosis. HP-treated blastocysts were processed for gene expression analysis (AQP3, ATP1A1, BAX, CDH1, HSP90β, NANOG, OCT4 and TP53) 1, 5 h after the end of HP exposure. Results showed that the hatching rate of embryos treated at 40 MPa was significantly higher than that of the 60 MPa-treated group (P < 0.01) and similar to untreated embryos. Blastocysts exposed at 40 MPa showed higher ICM (P < 0.05) and TE (P < 0.01) cell number and a lower percentage of picnotic nuclei (P < 0.05) compared with the control group. Significantly lower abundance for BAX (P < 0.01) and OCT4 (P < 0.05) transcripts were observed in HP embryos than in the control group. In conclusion, treatment with HP improved the quality of in vitro-produced ovine blastocysts by increasing their cell number and reducing the proportion of nuclear picnosis.

scholarly journals Dnmt3l-knockout donor cells improve somatic cell nuclear transfer reprogramming efficiency

Reproduction ◽  
2015 ◽  
Vol 150 (4) ◽  
pp. 245-256 ◽  
Author(s):  
Hung-Fu Liao ◽  
Chu-Fan Mo ◽  
Shinn-Chih Wu ◽  
Dai-Han Cheng ◽  
Chih-Yun Yu ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Dna Methyltransferase ◽  
Inner Cell Mass ◽  
Trichostatin A ◽  
Cell Mass ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Oct4 Expression ◽  
Donor Cells

Nuclear transfer (NT) is a technique used to investigate the development and reprogramming potential of a single cell. DNA methyltransferase-3-like, which has been characterized as a repressive transcriptional regulator, is expressed in naturally fertilized egg and morula/blastocyst at pre-implantation stages. In this study, we demonstrate that the use of Dnmt3l-knockout (Dnmt3l-KO) donor cells in combination with Trichostatin A treatment improved the developmental efficiency and quality of the cloned embryos. Compared with the WT group, Dnmt3l-KO donor cell-derived cloned embryos exhibited increased cell numbers as well as restricted OCT4 expression in the inner cell mass (ICM) and silencing of transposable elements at the blastocyst stage. In addition, our results indicate that zygotic Dnmt3l is dispensable for cloned embryo development at pre-implantation stages. In Dnmt3l-KO mouse embryonic fibroblasts, we observed reduced nuclear localization of HDAC1, increased levels of the active histone mark H3K27ac and decreased accumulation of the repressive histone marks H3K27me3 and H3K9me3, suggesting that Dnmt3l-KO donor cells may offer a more permissive epigenetic state that is beneficial for NT reprogramming.

194 Extracellular vesicles derived from ampullary oviductal fluid improve developmental competence of bovine oocytes

2020 ◽  
Vol 32 (2) ◽  
pp. 225
Author(s):  
A. Asaadi ◽  
K. Pavani ◽  
N. Azari Dolatabadi ◽  
P. Van Damme ◽  
A. Van Soom
Keyword(s):  
Extracellular Vesicles ◽  
Follicular Fluid ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Control Group ◽  
Cleavage Rate ◽  
Blastocyst Quality ◽  
Oviductal Fluid ◽  
Bovine Oocytes

Invivo, oocytes mature in the presence of follicular fluid (FF) and ampullary oviductal fluid (AOF), starting from premature to final maturation stages (3-6h postovulation). Extracellular vesicles (EVs) have been identified as important mediators of gamete/embryo-maternal interactions. They carry regulatory molecules, such as microRNAs and mRNAs. So far, the functionality of EVs derived from FF and AOF has not yet been determined. The aim of this study was to evaluate the effect of EVs derived from FF and AOF during invitro oocyte maturation (IVM) on the development and quality of resulting bovine embryos. Follicular fluid of a preovulatory follicle was collected by ovum pickup, and AOF was collected from the oviducts of slaughtered cows in early luteal phase. Then, EVs from FF and AOF were isolated by size exclusion chromatography and confirmed by western blot. The concentration of EVs was determined by NanoDrop and Nanoparticle tracking analysis. Integrity and size of EVs were assessed by electron microscopy. Bovine oocytes (n=1331) were allocated at random to five groups. In the control group (C), oocytes (n=347) were cultured for 22.5h in maturation medium (TCM-199 and epidermal growth factor 20ngmL−1) (MM). In the negative control group (NC), oocytes (n=331) were cultured for 18h in MM, and then transferred into fresh MM for 4.5h. In the FF group, oocytes (n=162) were cultured in MM supplemented with FF EVs (12.5µgmL−1) for 18h, and then transferred to MM for 4.5h. In the AOF group, oocytes (n=328) were cultured for 18h in EV-free MM, and then transferred to MM supplemented with AOF EVs (1.7µgmL−1) for 4.5h. In the FF+AOF group, oocytes (n=163) were cultured in MM supplemented with FF EVs for 18h, and then transferred into MM supplemented with AOF EVs for 4.5h. At 22.5h post-incubation, mature oocytes were fertilized invitro. At 21h post-insemination, presumptive zygotes were denuded and cultured in synthetic oviductal fluid with insulin-transferrin-selenium for 8 days. Cleavage rate and blastocyst rate were recorded at 48h and 8 days post-insemination, respectively. Blastocyst quality was assessed by differential apoptotic staining. The data were analysed using one-way ANOVA. Cleavage rate was not affected by the treatment, whereas blastocyst yield in the AOF group (48.75%) was significantly higher than that in the C (42.91%) and NC (37.72%) groups. In addition, embryos produced in the AOF group showed the highest number of trophectoderm cells and inner cell mass cells, and lowest number of apoptotic cells (P&lt;0.05). Trophectoderm and inner cell mass cells were higher and apoptotic cells were lower in FF and FF+AOF compared with the C and NC groups. In conclusion, adding AOF EVs to oocyte MM in the last 4.5h of IVM improves quantity and quality of blastocysts. In addition, although FF EVs showed no effect on blastocyst yield, they improved blastocyst quality by increasing cell number.

Quality of rabbit vitrified/thawed transgenic embryos

Zygote ◽  
2011 ◽  
Vol 21 (1) ◽  
pp. 53-58 ◽  
Author(s):  
P. Chrenek ◽  
Z. Turanová ◽  
J. Slamečka ◽  
A. V. Makarevich
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Developmental Rate ◽  
Control Group ◽  
Cell Counts ◽  
Transgenic Rabbits ◽  
Significant Difference ◽  
Transgenic Embryos ◽  
Rabbit Embryos

SummaryThe aim of our study was to investigate the influence of vitrification on developmental rate and quality (total number of cells, number of blastomeres in inner cell mass (ICM) area, apoptotic index and embryo diameter) of transgenic (carrying an endogenous–hFVIII or exogenous–enhanced green fluorescent protein (EGFP) gene) rabbit embryos. EGFP-positive rabbit embryos were produced under in vitro conditions by the microinjection of foreign genes into the pronucleus of fertilized eggs. The transgenic rabbit embryos with the hFVIII gene were produced by mating homozygous transgenic rabbits and flushing at the single-cell stage. Developmental rate of vitrified/thawed transgenic embryos that reached hatching blastocyst stage (68.00% and 69.00%) and differed significantly (p < 0.001) from those in control embryos (100.00%). Significant difference (p < 0.05) was found in total cell counts between control (117.00 ± 36.00) and vitrified (141.00 ± 34.80) hFVIII-positive embryos. The higher proportion of ICM cells (32.00%) and greatest embryo diameter (130.85 ± 10.90) were found in the control group compared with the transgenic. Ratio of apoptotic cells was significantly higher (p < 0.01) in the control group (2.50%) and vitrified EGFP-positive embryos (2.90%) compared with the vitrified, hFVIII-positive group of embryos (0.70%). Our results demonstrate that neither gene microinjection itself, nor exogenous (EGFP) and endogenous (hFVIII) gene expression interferes with developmental rate and quality of rabbit embryos. However, a combination of microinjection and vitrification significantly decreases (p < 0.001) the survival rate of rabbit embryos.

Maternal influence on oocyte collection and embryo development after transvaginal puncture and in vitro fertilisation in dairy cows

2001 ◽  
Vol 26 (2) ◽  
pp. 433-435
Author(s):  
S. Chastant-Maillard ◽  
H. Quinton ◽  
C. Douar ◽  
J. Marchal ◽  
C. Richard ◽  
...  
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Formation Rate ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Cell Counting ◽  
In Vitro Fertilisation ◽  
Oocyte Collection

AbstractThe aim of the study was to evaluate differences between cows in the “quality” of their oocytes defined as their ability to support embryonic development. Ten cows from the same herd, all primiparous and non-pregnant were submitted for oocyte collection by Ovum-Pick Up (OPU). The oocytes were matured in vitro and fertilised with semen from the same bull. In vitro embryo development, both quantitatively (percentages showing cleavage and forming blastocysts) and qualitatively (differential cell counting in blastocysts) was determined at the blastocyst stage (Day 7). The number of oocytes collected, the number of blastocysts obtained and the blastocyst formation rate varied between cows (P<0.001). The mean percentage of inner cell mass cells tended to higher for embryos derived from one cow. These results provide evidence that the quality of the oocytes was influenced by their maternal origin. Follicular growth also varied between cows.

scholarly journals 165RE-EXPANSION AND QUALITY OF SPLIT BOVINE EMBRYOS IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 205 ◽  
Author(s):  
P. Kesseler ◽  
E. Mahabir ◽  
M. Köster ◽  
M. Gilles ◽  
K. Wimmers ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Counting ◽  
Bovine Embryos ◽  
Becton Dickinson ◽  
Group A ◽  
Group B ◽  
Number Of Cells

Transferring split bovine embryos results in a higher number of calves born per embryo. In addition to generating genetically identical progeny, biopsies can be made for molecular biological analyses. We aimed to determine the effect of splitting ratio on the in vitro development of Day 7 (164 to 168h post insemination) IVP bovine embryos. The inner cell mass (ICM) and trophoblast cells were split in three ratios (50:50, 60:40 and 70:30) with a Beaver microblade (Becton Dickinson, N.J., USA.) fixed to a micromanipulator under an inverse microscope at 100 X (Leica, Bensheim, Germany). Split blastocysts were cultured singly in 50μL drops of CR1aa medium at 39°C under 5% CO2 in a moisture-saturated atmosphere. After 1 and 2h culture, the morphology was assessed by judging the shape of the embryos and re-development of the blastocoel. On Day 8 (after 22h culture), the shape of the blastocysts, development of the ICM, blastocoel, proportion of degenerated cells and embryos and re-expanded blastocysts were recorded. Embryos were stained with propidium iodide and Hoechst 33258 for cell counting. The re-expansion status of Group A (50, 60 and 70%) and B (50, 40 and 30%) embryos after 1 and 2h and their quality after 22h culture (1: excellent=&lt;10% degenerated cells, well-defined ICM; 2: fair=&lt;20% degenerated cells) are shown in Table 1. With regards to Group A split blastocysts, a higher (P&lt;0.05) percentage of embryos that re-expanded after 1 and 2h and which yielded Quality 1 and 2 embryos suitable for embryo transfer was observed with the 60% and 70% than with demi-embryos. There were significant differences (P&lt;0.05) between all split blastocysts in Group B after 1h culture. The 30% split embryos showed the lowest re-expansion rate and quality of embryos after 2h and 22h culture, respectively. No differences (P&lt;0.05) were seen in the ratio of the ICM to the total number of cells in both Group A and B. This study showed that the ratio in which blastocysts were split had a significant effect on re-expansion and quality but not on the number of cells. Table 1

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