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.

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.

scholarly journals Vitrification of In Vitro Produced Porcine Blastocysts: Influence of Cryoprotectants Toxicity and Embryo Age

2019 ◽  
Vol 47 (1) ◽  
Author(s):  
Lain Uriel Ohlweiler ◽  
Joana Claudia Mezzalira ◽  
Alceu Mezzalira
Keyword(s):  
Cell Number ◽  
Lower Percentage ◽  
Toxicity Tests ◽  
Hatching Rate ◽  
Embryo Viability ◽  
Embryo Survival ◽  
Porcine Embryo ◽  
Negative Role ◽  
Cryoprotectant Solution

Background: Porcine embryos are sensible to all assisted reproduction manipulations, especially the ones that involve cryopreservation. Despite the high cryoprotectant concentrations routinely applied, vitrification is the most effective technique to date. These substances toxicity can also play a negative role in embryo viability. During in vitro porcine embryo production, the speed of development is often unevenly distributed. It is possible that their development speed, affects embryo tolerance to cryoprotectants. This study aimed to evaluate the toxicity of porcine embryos of days 5 or 6 of culture to cryoprotectant agents; as well as to assess embryo survival to vitrification.Material, Methods & Results: Parthenogenetic porcine blastocysts and expanded blastocysts of days 5 and 6 of culture were exposed to toxicity tests (experiments 1 and 2) and vitrification (experiment 3) using different protocols. In the first experiment, three different cryoprotectants were used (Dimethyl sulfoxide - DMSO, Ethylene glycol – EG, and Sucrose - SUC), combined in three different associations (G1: 15% EG + 15% DMSO with 0.5M SUC; G2: 16% EG + 16% DMSO with 0.4M SUC; G3: 18% EG + 18% DMSO with 0.5M SUC). In the fresh Control, embryos of day 6 are more sensible than the ones of day 5, whom showed a lower hatching rate (39.7 vs. 60.8%). After the toxicity (Experiment 1) test, the G1 showed better expansion rates in day 6 (50.0 vs 31.0 and 3.6% for G2 and G3) and higher hatching of day 6 compared to G2 and G3 (23.2, vs. 8.6 and 0.0% for G2 and G3). The fresh non hatched embryos at day 8, derived at day 6, had a lower percentage of cells with cleaved caspase-3 (20.2%) compared with the G1 (30.5%), G2 (31.4%) and G3 (30.5%). The hatched embryos of day 5 from G2 had lower total cell number (TCN) compared with the day 6 hatched embryos, whereas in G1 the TCN was not affected. The second experiment compared EG combined to one of these three extracellular cryoprotectants: Polyvinylpyrrolidone/sucrose/trehalose (respectively groups: PVP, SUC, TRE). The group SUC has raised the best results for day 5 embryos, whereas for day 6 embryos SUC and TRE were both best. The third experiment tested four vitrification protocols, being P1: EG+DMSO+TRE/warming with SUC; P2: EG+DMSO+TRE/warming TRE; P3: EG+TRE/ warming SUC; P4: EG+TRE/warming TRE. The expansion of vitrified day 5 embryos was higher in the P1 (20.0%) in comparison with the other three groups (4.3, 4.3 and 4.4% for P2, P3 and P4, respectively), with no difference for their hatching rates, been it lower comparing to the Control. Day 6 embryos showed no difference in expansion and hatching for the vitrified groups, been them lower than the Control.Discussion: Embryos obtained on day 6 are more sensible than the ones of day 5, fact observed when the embryos were exposed to cryoprotectant solution, as well by the behavior of the no treated Control embryos. The toxicity increases as it does the concentration of intracellular cryoprotectant, where over 16% of the intracellular cryoprotectors already affected the day 6 embryos development. For the day 5 embryos however, 15 or 16% of the intracellular cryoptrotectors, had similar behavior to the embryos. For the extracellular solutions, however, it is variable according the embryos development speed. Indeed, it is necessary to adjust the cryoprotectors to be used to cryopreserve porcine in vitro produced embryos obtained at days 5 and 6 of culture.

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.

87 HIGH HYDROSTATIC PRESSURE TREATMENT IMPROVES THE QUALITY OF IN VITRO-PRODUCED OVINE BLASTOCYSTS

2010 ◽  
Vol 22 (1) ◽  
pp. 202 ◽  
Author(s):  
L. Bogliolo ◽  
F. Ariu ◽  
S. Uccheddu ◽  
A. Strina ◽  
I. Rosati ◽  
...  
Keyword(s):  
Hydrostatic Pressure ◽  
Beneficial Effect ◽  
High Hydrostatic Pressure ◽  
Cell Number ◽  
Embryo Cryopreservation ◽  
Hatching Rate ◽  
Pressure Treatment ◽  
Custom Made

High hydrostatic pressure (HHP) has been introduced into the field of embryology recently. It has been reported that a sublethal HHP could induce the synthesis of molecular chaperons to protect the embryos from other environmental stresses. The possible beneficial effect of HHP was examined on embryo vitrification, and improved cryotolerance has been achieved in HHP-treated mouse and bovine embryos. In the present study we evaluated, first, the behavior of in vitro-produced ovine blastocysts after treatment with 2 different HHP conditions. In the second part of the study, optimized pressure treatment was combined with a cryopreservation protocol. Day 7 in vitro-produced ovine blastocysts were loaded in 0.5-mL straws and pressure treated in a custom-made hydrostatic pressure chamber (Cryo-Innovation Ltd., Budapest, Hungary) according to the following conditions: -60 MPa, 70 min, 38°C; and -40 MPa, 70 min, 38°C. After HHP treatment, embryos were cultured for 24 h and their survival evaluated upon morphological appearance, re-expansion of the blastocoel, and hatching from the zona pellucida. Blastocysts were fixed and stained, observed with confocal microscope to evaluate blastocyst cell number, and mitotic and picnotic index. Untreated blastocysts were used as controls. On the basis of results obtained in this experiment, groups of blastocysts were exposed to 40 MPa for 70 min at 38°C and, following 2 h incubation after HHp treatment, were vitrified using cryotops. After warming, embryos were checked for re-expansion and hatching and finally fixed and stained as described for fresh embryos. Vitrified untreated blastocysts were used as controls. Results showed that the hatching rate of embryos treated at 40 MPa was significantly higher than that of 60 MPa-treated group (87.5 v. 0%; P < 0.01, chi-squared test) and similar to that of control embryos (85.3%) after 24 h culture. Blastocysts exposed at 40 MPa showed higher cell number compared with control embryos (161.3 ± 8.7 v. 123.9 ± 9.4, P < 0.01, ANOVA ± SD) and a lower picnotic index (PI: 1.3 ± 0.4% v. 3.8 ± 0.6%, P < 0.05). Similar re-expansion rate has been recorded between HHP treated and untreated (86.6 v. 89.6%) vitrified blastocysts as well as hatching rates (53.3 v. 48.3) and embryo cell number (131 ± 4.6 v. 139 ± 5.8). Conversely, HHP treatment significantly decreased picnotic index of vitrified treated embryos compared with control (2.3 ± 0.9 v. 4.8 ± 0.5, P < 0.05). We concluded that HHP treatment significantly improved the quality of in vitro-produced ovine blastocysts by increasing their cell number and reducing the proportion of picnosis. The HHP treatment exerted a positive effect in vitrified blastocysts, decreasing the number of picnotic nuclei. Further studies are needed to evaluate the possible beneficial effect at the molecular level of HHP treatment after vitrification and also to test if different interval times between HHP treatment and embryo cryopreservation could affect embryo response.

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.

The human blastocyst: cell number, death and allocation during late preimplantation development in vitro

Development ◽  
1989 ◽  
Vol 107 (3) ◽  
pp. 597-604 ◽  
Author(s):  
K. Hardy ◽  
A.H. Handyside ◽  
R.M. Winston
Keyword(s):  
Cell Death ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Cell Numbers ◽  
Human Blastocyst ◽  
Development In Vitro

The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/− 8.1 cells, which increased to 84.4 +/− 5.7 and 125.5 +/− 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/− 6.0 and 40.3 +/− 5.0, respectively) and then doubled on day 7 (80.6 +/− 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/− 4.0 and 41.9 +/− 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/− 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.

Abnormal development of pre-implantation mouse embryos grown in vitro with [3H]thymidine

Development ◽  
1973 ◽  
Vol 29 (3) ◽  
pp. 601-615
Author(s):  
M. H. L. Snow
Keyword(s):  
Specific Activity ◽  
Inner Cell Mass ◽  
Cell Damage ◽  
Cell Mass ◽  
Cell Number ◽  
Mouse Embryos ◽  
Abnormal Development ◽  
Tritium Concentration ◽  
Cell Stage

Mouse embryos were grown in vitro from the 2-cell stage to blastocysts in the presence of [3H]thymidine. Methyl-T-thymidine and thymidine-6-T(n) were used and both forms found to be lethal at concentrations above 0·1 μCi/ml. Both forms of [3H]Tdr at concentrations between 0·01 and 0·1 μCi/ml caused a highly significant (P &lt; 0·001) reduction in blastocyst cell number. The reduction in cell number, which was positively correlated with specific activity and tritium concentration, was associated with cell damage typical of radiation damage caused by tritium disintegration. Thymidine-6-T(n) also significantly reduced the number of 2-cell embryos forming blastocysts whereas methyl-T-Tdr did not. This difference in effect is assumed to be caused by contamination of one form of [3H]Tdr with a by-product of the tritiation process. A study of the cleavage stages showed that almost all the reduction in cell numbers could be accounted for by selective cell death occurring at the 16-cell stage. Cells which survive that stage cleave at a normal rate. The cells that are most susceptible to [3H]Tdr damage were found to normally contribute to the inner cell mass. The [3H]Tdr-resistant cells form the trophoblast. It is possible to grow blastocysts in [3H]Tdr such that they contain no inner cell mass but are composed entirely of trophoblast. Comparatively short (12 h) incubation with [3H]Tdr at any stage prior to the 16-cell stage will cause this damage. Possible reasons for this differential effect are discussed, and also compared with damage caused by X-irradiation.

scholarly journals Simultaneous gene quantitation of multiple genes in individual bovine nuclear transfer blastocysts

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 231-242 ◽  
Author(s):  
Craig Smith ◽  
Debbie Berg ◽  
Sue Beaumont ◽  
Neil T Standley ◽  
David N Wells ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Ectopic Expression ◽  
Cell Number ◽  
Transcript Levels ◽  
Multiple Genes

During somatic cell nuclear transfer (NT), the transcriptional status of the donor cell has to be reprogrammed to reflect that of an embryo. We analysed the accuracy of this process by comparing transcript levels of four developmentally important genes (Oct4,Otx2,Ifitm3,GATA6), a gene involved in epigenetic regulation (Dnmt3a) and three housekeeping genes (β-actin, β-tubulinandGAPDH) in 21 NT blastocysts with that in genetically half-identicalin vitroproduced (IVP,n=19) andin vivo(n=15) bovine embryos. We have optimised an RNA-isolation and SYBR-green-based real-time RT-PCR procedure allowing the reproducible absolute quantification of multiple genes from a single blastocyst. Our data indicated that transcript levels did not differ significantly between stage and grade-matched zona-free NT and IVP embryos except for Ifitm3/Fragilis, which was expressed at twofold higher levels in NT blastocysts.Ifitm3expression is confined to the inner cell mass at day 7 blastocysts and to the epiblast in day 14 embryos. No ectopic expression in the trophectoderm was seen in NT embryos. Gene expression in NTand IVP embryos increased between two- and threefold for all eight genes from early to late blastocyst stages. This increase exceeded the increase in cell number over this time period indicating an increase in transcript number per cell. Embryo quality (morphological grading) was correlated to cell number for NT and IVP embryos with grade 3 blastocysts containing 30% fewer cells. However, only NT embryos displayed a significant reduction in gene expression (50%) with loss of quality. Variability in gene expression levels was not significantly different in NT, IVP orin vivoembryos but differed among genes, suggesting that the stringency of regulation is intrinsic to a gene and not affected by culture or nuclear transfer.Oct4levels exhibited the lowest variability. Analysing the total variability of all eight genes for individual embryos revealed thatin vivoembryos resembled each other much more than did NT and IVP blastocysts. Furthermore,in vivoembryos, consisting of 1.5-fold more cells, generally contained two- to fourfold more transcripts for the eight genes than did their cultured counterparts. Thus, culture conditions (in vivoversusin vitro) have greater effects on gene expression than does nuclear transfer when minimising genetic heterogeneity.

15 HISTONE ACETYLATION PROFILE OF PORCINE EMBRYOS PRODUCED BY 2 CLONING METHODS WITH OR WITHOUT IN VITRO CULTURE

2016 ◽  
Vol 28 (2) ◽  
pp. 137
Author(s):  
Y. Liu ◽  
A. Lucas-Hahn ◽  
B. Petersen ◽  
R. Li ◽  
D. Hermann ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Histone Acetylation ◽  
Mrna Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Cell Numbers ◽  
Cloning Method

Conventional “Dolly”-based cloned (CNT) embryos maintain zona pellucida and can be transferred early in development. Handmade cloned (HMC) embryos are zona free and are cultured to later stages for transfer. We have shown differences between HMC and CNT embryos (Rep. Fert. Dev. 26, 123), and both in vitro culture and cloning method (NT) are associated with alterations in histone acetylation. More studies are needed to clarify whether CNT and HMC embryos differ in epigenetic profiles due to NT method or culture condition. Here we investigated histone acetylation profile of NT embryos produced by CNT or HMC with or without 5 to 6 days in vitro culture, emphasising quality and gene expression in resulting embryos. Both NT methods were performed on Day 0 (D0) with same oocyte batch, donor cells, and culture medium (CNT in group, HMC in well of well). On D0, 5, and 6 after CNT (Clon. Stem Cells 10, 355) or HMC (Zygote 20, 61), all developed embryos of all morphological qualities were collected for immunostaining of H3K18ac, and on D0 and 6 for mRNA expression of the genes KAT2A/2B, EP300, HDAC1/2, DNMT1o/s, and GAPDH. Embryo quality was evaluated normal (clear inner cell mass, high cell number, no fragments) or bad (no clear inner cell mass, low cell number, fragments). Cell numbers per blastocyst were counted on D5 and 6. Differences in cell number and H3K18ac level between different groups and days were analysed by ANOVA; gene expression data were analysed by GLM (SAS version 9.3, SAS Institute Inc., Cary, NC, USA). Embryo development rates of both NT methods were reported previously (Rep. Fert. Dev. 26, 123). On D5 and 6, all HMC embryos were evaluated as normal, but the CNT group contained both normal and bad embryos. Regarding cell numbers (Table 1), on D5 there was no difference between normal CNT and HMC embryos, but numbers were lower in CNT bad embryos. On D6 the blastocyst cell number was lower in both normal and bad CNT embryos compared with HMC. Regarding H3K18ac levels (Table 1), no differences were found on D5 between normal CNT and HMC embryos, but on D6 both CNT normal and bad embryos had higher H3K18ac level compared with HMC. On D0, no difference was found in mRNA expression of all 8 genes. On D6, KAT2A expression was slight increased (1.8-fold) in CNT compared with HMC embryos (P < 0.05). In conclusion, no differences were found between CNT and HMC embryos after completed NT procedure (D0) or after 5 days in vitro culture. However, differences in quality (cell number and H3K18ac) and gene expression between the 2 NT methods were observed when blastocyst expansion was initiated (D6). Thus, the 2 NT methods seem to produce embryos of similar quality, which is maintained over 5 days in vitro culture, but thereafter gene expression and histone acetylation are more active in CNT embryos. Table 1.Cell number and H3K18ac level1

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