Vitrification of bovine blastocysts pretreated with sublethal hydrostatic pressure stress: evaluation of post-thaw in vitro development and gene expression

2011 ◽  
Vol 23 (4) ◽  
pp. 585 ◽  
Author(s):  
E. Siqueira Filho ◽  
E. S. Caixeta ◽  
C. Pribenszky ◽  
M. Molnar ◽  
A. Horvath ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hydrostatic Pressure ◽  
Transcript Abundance ◽  
Developmental Competence ◽  
Hatching Rate ◽  
In Vitro Development ◽  
Stress Treatment ◽  
Pressure Stress ◽  
Vitro Development

Sublethal stress treatment has been reported to enhance gametes’ performance in subsequent procedures, such as cryopreservation. The aim of the present study was to evaluate the effect of different equilibration times between the termination of a sublethal hydrostatic pressure (HP) stress treatment and the initiation of vitrification on the post-thaw survival, continued in vitro development, hatching rate and gene expression of selected candidate genes of in vitro-produced (IVP) expanded bovine blastocysts. Day 7 IVP blastocysts were subjected to 600 bar pressure for 60 min at 32°C. Immediately after pressure treatment (HP0h) or after 1 or 2 h incubation (HP1h and HP2h groups, respectively), embryos were either vitrified and warmed using the open pulled straw method, followed by 72 h in vitro culture or were stored at –80°C until gene expression analysis. Re-expansion and hatching rates after vitrification–warming were significantly (P < 0.05) higher in the HP0h (88 and 76%, respectively) and HP1h (90 and 75%, respectively) groups than in the untreated (82 and 63%, respectively) and HP2h groups (79 and 70%, respectively). Moreover, the HP1h group showed further improvement in the speed of re-expansion and resumption of normal in vitro development. Cumulative analysis of all genes (SC4MOL, HSP1A1A, SOD2 and GPX4) revealed a similar pattern of expression, with a tendency for peak transcript abundance 1 h after HP treatment. Application of HP stress treatment was found to be efficient in increasing the in vitro developmental competence of vitrified bovine embryos.

Cells under pressure: how sublethal hydrostatic pressure stress treatment increases gametes' and embryos' performance

2011 ◽  
Vol 23 (1) ◽  
pp. 48 ◽  
Author(s):  
Csaba Pribenszky ◽  
Gabor Vajta
Keyword(s):  
Hydrostatic Pressure ◽  
Cell Survival ◽  
New Approach ◽  
In Vitro Development ◽  
Stress Treatment ◽  
Sublethal Stress ◽  
Minimal Damage ◽  
Pressure Stress ◽  
Vitro Development

The principal approach in in vitro embryo culture and manipulation has been a defensive one: procedures aim to satisfy passively the supposed or real physiological needs of gametes and embryos. Similarly, during cryopreservation the aim is to cause minimal damage to cells whilst attempting to obtain the highest achievable cell survival. However, carefully chosen and precisely controlled sublethal stress treatment of cells has been described to improve embryos’ and gametes’ performance, and, as a consequence, subsequent morphological survival, fertilisation, in vitro development, pregnancy and farrowing rates improved considerably compared with untreated controls. This review summarises studies that open up a new approach: instead of – and besides – trying to passively reduce the harm to cells during in vitro manipulations and culture, procedures may also prepare the cells themselves to ward off or reduce the damage by turning up the cells’ own, inner capacities.

89 IMPROVED POST-WARMING DEVELOPMENTAL COMPETENCE OF OPEN PULLED STRAW-VITRIFIED IN VITRO-PRODUCED BOVINE BLASTOCYSTS BY SUBLETHAL HYDROSTATIC PRESSURE PRETREATMENT

2008 ◽  
Vol 20 (1) ◽  
pp. 125 ◽  
Author(s):  
C. Pribenszky ◽  
F. E. Siqueira ◽  
M. Molnár ◽  
A. Harnos ◽  
R. Rumpf
Keyword(s):  
Hydrostatic Pressure ◽  
Developmental Competence ◽  
Equilibration Time ◽  
In Vitro Development ◽  
Custom Made ◽  
Boar Semen ◽  
Pre Treatment ◽  
Vitro Development ◽  
Hatching Rates

Vitrification of in vitro (IVP)-produced bovine blastocysts is well established, reaching post-warming hatching rates close to 70–80% in vitro. Nevertheless, improvements still are needed regarding realizable pregnancy rates. High hydrostatic pressure (HHP) treatment of fresh boar semen before freezing increased the litter size achieved by insemination of frozen–thawed boar semen (Kuo et al. 2007 6th Int. Conf. Boar Semen Pres, Alliston, ON, Canada, poster #22); HHP treatment-related improvements were also observed in the in vitro cryosurvival of mouse blastocysts (Pribenszky et al. 2004 Reprod. Fertil. Dev. 17, 199–200), bull and boar semen, and pig oocytes, theoretically by the sublethal stress-induced production/stabilization of shock proteins (Pribenszky et al. 2006 Reprod. Fertil. Dev. 18, 162–163; 2007 Reprod. Fertil. Dev. 19, 181–182; and b; Du et al. 2007). The aim of the present study was to improve the post-warming in vitro developmental competence of vitrified bovine IVP blastocysts through the application of HHP as pre-treatment, in order to apply the protocol in later in vivo experiments. Day 7 IVP blastocysts were aspirated in TQC holding medium (AB Technology, Sao Paulo, Brazil) into 0.25-mL straws. Straws were pressure-treated in a custom-made hydrostatic pressure chamber (Cryo-Innovation Ltd., Budapest, Hungary), using water as pressure medium. Six hundred bar pressure was applied for 60 min at 32�C. Immediately after pressure treatment, or following 60- or 120-min incubation, embryos were vitrified and warmed using open pulled straws (OPS) according to the method of Vajta et al. (1998 Mol. Reprod. Dev. 51, 53–58). Untreated blastocysts were vitrified as controls. After warming, embryos were cultured in vitro in SOF (Holm et al. 1999 Theriogenology 52, 683–700) for 72 h. Embryos were checked for re-expansion and hatching at 4, 24, 48, and 72 h post-warming. For the experiment, 404 blastocysts were used in 5 replicates. Logistic regression was used for statistical evaluation. All vitrified groups were inferior compared to the non-vitrified control (97%, 97%, 98, and 100% expansion; 0%, 23%, 72 and 91% hatching at 4, 24, 48, and 72 h, respectively). HHP treatment had a significant effect (P > 0.05) on the post-warming developmental competence of vitrified blastocysts. HHP treatment followed by 60 min of equilibration proved to be superior among all treatment groups regarding both re-expansion and hatching rates and the speed of resumption of normal in vitro development (HHP treatment followed by a 60-min equilibration time before vitrification/warming: re-expansion rates: 88%, 89%, 85, and 90%; hatching rates: 0%, 22%, 51, and 73% v. non-treated vitrified/warmed controls: re-expansion rates: 63%, 69%, 71, and 81%; hatching rates: 0%, 6%, 43, and 63%; at 4, 24, 48, and 72 h post-warm, respectively). In conclusion, hydrostatic pressure pre-treatment significantly improved in vitro survival and hatching rates as well as the speed of resumption of normal in vitro development. Further studies are needed to reveal the molecular-biological implications of the HHP treatments, as well as field trials to test if the in vitro improvements can be confirmed by pregnancy and birth rates. This work was supported by EMBRAPA and a Kozma grant, Hungary.

Effects of the HDAC inhibitor scriptaid on the in vitro development of bovine embryos and on imprinting gene expression levels

2018 ◽  
Vol 110 ◽  
pp. 79-85 ◽  
Author(s):  
R. Laguna-Barraza ◽  
M.J. Sánchez-Calabuig ◽  
A. Gutiérrez-Adán ◽  
D. Rizos ◽  
S. Pérez-Cerezales
Keyword(s):  
Gene Expression ◽  
Hdac Inhibitor ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Expression Levels ◽  
Imprinting Gene ◽  
Vitro Development ◽  
Gene Expression Levels

64 VALPROIC ACID ENHANCES IN VITRO DEVELOPMENT OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS IN PIGS

2010 ◽  
Vol 22 (1) ◽  
pp. 190
Author(s):  
Y. J. Kim ◽  
K. S. Ahn ◽  
M. J. Kim ◽  
H. Shim
Keyword(s):  
Stem Cells ◽  
Valproic Acid ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Developmental Competence ◽  
Control Group ◽  
In Vitro Development ◽  
Vitro Development ◽  
And Control

Epigenetic modification influences reprogramming and subsequent development of somatic cell nuclear transfer embryos. Such modification includes an increase of histone acetylation and a decrease of DNA methylation in transferred donor nuclei. Histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) and valproic acid (VPA) have been known to maintain high cellular levels of histone acetylation. Hence, the treatment of HDACi to NT embryos may increase efficiency of cloning. Indeed, TSA treatment has significantly enhanced the developmental competence of nuclear transfer embryos in several species including pigs (Zhang et al. 2007 Cloning Stem Cells 9, 357-363; Li et al. 2008 Theriogenology 70, 800-808). Valproic acid, another type of HDACi, has often been used to assist reprogramming of somatic cells into induced pluripotent stem cells in mice. In the present study, we tested the potency of VPA compared with TSA on the enhancement of in vitro development in porcine nuclear transfer embryos. Reconstructed embryos were produced by transferring nuclei of adult ear skin fibroblasts into enucleated oocytes. After electrical activation, these embryos were cultured in PZM-3 containing no HDACi (control), 5 mM VPA, or 50 nM TSA for 24 h, and another 5 days thereafter without HDACi. At least 3 replicates were conducted for the following experiments. The rates of cleavage were not different among the VPA, TSA, and control groups. However, the rate of blastocyst development was significantly higher (P < 0.05) in embryos treated with VPA than in those treated with TSA and without HDACi (125/306, 40.8% v. 94/313, 30.0% v. 80/329, 24.3%). Differential staining of inner cell mass (ICM) and trophectoderm (TE) also supported the beneficial effect of VPA treatment in NT embryos. Compared with the control group, the number of TE cells was significantly increased (P < 0.05) in the VPA and TSA treatment groups (79.3 ± 7.4 v. 74.6 ± 9.2 v. 40.0 ± 6.7). Moreover, VPA treatment significantly increased (P < 0.05) the number of ICM cells compared with the control (15.6 ± 1.7 v. 10.8 ± 2.6), whereas no differences were observed between the TSA treatment and control group (12.9 ± 3.0 v. 10.8 ± 2.6). The present study demonstrates that VPA enhances in vitro development of nuclear transfer embryos, in particular by an increase of blastocyst formation and the number of ICM cells, suggesting that VPA may be more potent than TSA in supporting developmental competence of cloned embryos. However, long-term effects of different HDACi in the development of nuclear transfer embryos, including any adverse outcome from destabilizing epigenetic condition, remain to be determined by further in vivo embryo transfer studies.

scholarly journals 205.The effect of FSH concentration during IVM and gamete co-incubation length during IVF on the development of unstimulated prepubertal ewe oocytes

2004 ◽  
Vol 16 (9) ◽  
pp. 205 ◽  
Author(s):  
K. M. Morton ◽  
W. M. C. Maxwell ◽  
G. Evans
Keyword(s):  
Blastocyst Stage ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
In Vitro Development ◽  
Oocyte Collection ◽  
Chi Squared ◽  
Development In Vitro ◽  
Stage 1 ◽  
Vitro Development

The developmental competence of prepubertal oocytes can be increased by the administration of gonadotrophins prior to oocyte collection (1); but this is not possible with abattoir-sourced oocytes, and modifications to the IVP system may increase in vitro development. Experiments were conducted to determine the effects of FSH concentration (10, 20 or 60 μg mL-1) during IVM (5 replicates) and gamete co-incubation length (short: 2-3 h, long: 18-20 h) during IVF (6 replicates) on subsequent embryonic development. For both experiments ovaries were collected from prepubertal lambs (16-24 weeks) slaughtered at an abattoir and embryos produced in vitro (1). Data were analysed by chi-squared test. Oocyte cleavage at 48 hours post-insemination (hpi) was higher for oocytes matured in medium containing 20 (60/77; 77.9%) and 60 (56/73; 76.7%) than 10 μg mL-1 (40/67; 59.7%) FSH. Blastocyst formation (% cultured oocytes) on Day 7 (Day 0 = IVF) was higher for oocytes matured with 20 (31/77; 40.3%) than 10 (16/67; 23.9%) or 60 μg mL-1 (20/73; 27.4%). Oocyte cleavage at 48 hpi was reduced for short (36/57; 63.2%) compared with long (49/55; 89.1%) co-incubation, although blastocyst formation (% cultured oocytes; Day 7) did not differ between groups (22/57; 38.6% and 23/55; 41.8%, respectively). These results demonstrate that increasing the FSH concentration above normal levels during IVM of prepubertal lamb oocytes improves development in vitro. Gamete co-incubation length did not influence the proportion of oocytes progressing to the blastocyst stage. (1) Morton et al. (2003) Proc. Soc. Reprod. Fert. P18.

267 TARGETED SUPPRESSION OF THE EXPRESSION OF MATERNAL AND EMBRYONIC GENES DURING IN VITRO DEVELOPMENT OF BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 241
Author(s):  
D. Tesfaye ◽  
K. Nganvongpanit ◽  
F. Rings ◽  
M. Gilles ◽  
D. Jennen ◽  
...  
Keyword(s):  
Free Water ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
First Polar Body ◽  
Vitro Development ◽  
Bovine Oocytes ◽  
E Cadherin

Despite enormous advances in the identification and sequencing of developmentally relevant bovine genes, the function of the majority of these transcripts is not yet known. Here we aimed to apply the RNA interference (RNAi) approach to suppress the expression of the maternal transcript c-mos (AY630920) and embryonic transcripts E-cadherin (AY508164) and Oct-4 (AY490804) during in vitro development of bovine embryos using microinjection of sequence-specific double-stranded RNA (dsRNA). For this 435-, 341- and 341-bp-long dsRNA specific to the coding sequences of c-mos, E-cadherin and Oct-4 transcripts, respectively, were synthesized using Promega RiboMax" T7 system (Promega, Madison, WI, USA), where sense and antisense strands were transcribed from the target DNA template. Slaughterhouse ovaries were used to aspirate bovine oocytes, which were matured in TCM-199 with 12% estrus cow serum (ECS), fertilized in Fert-TALP, and cultured in CR1 medium at 39�C under humidified atmosphere of 5% CO2 in air. In Experiment 1, immature oocytes were categorized into three groups, each containing 50-60 oocytes: those injected with c-mos dsRNA, those injected with RNase-free water, and uninjected controls. In Experiment 2, zygotes were categorized into four groups, each containing 50-60 zygotes: those injected with E-cadherin dsRNA, those injected with Oct-4 dsRNA, those injected with RNase-free water, and uninjected controls. Each experiment was repeated four times. The effect of dsRNA on in vitro development of oocytes or embryos was assessed after microinjection during culture. The level of mRNA and protein expression was investigated using real-time PCR and western blot analysis, respectively. Data were analyzed using SAS, version 8 (SAS Institute Inc., Cary, NC, USA). Microinjection of c-mos dsRNA resulted in a 70% reduction of c-mos transcript abundance after maturation compared to the water-injected and uninjected controls (P < 0.05). Similarly, microinjection of E-cadherin and Oct-4 dsRNA at the zygote stage resulted in 80% and 60% reduction in transcript abundance at the blastocyst stage, respectively, compared to the uninjected controls (P < 0.05). Decreases in the c-mos (39 kDa) and E-cadherin proteins (119 kDa) were observed in the c-mos and E-cadherin dsRNA-injected groups, respectively, compared to the control. A higher proportion of oocytes (75%) showed first polar body extrusion after maturation in c-mos dsRNA-injected groups, compared to 52% in water-injected and 57% in uninjected controls. Only 22% from E-cadherin dsRNA- and 24% from Oct-4 dsRNA-injected zygotes developed to the blastocyst stage compared to 39 and 37% blastocyst rates in water-injected and uninjected control groups, respectively. In conclusion, injection of sequence-specific dsRNA in bovine oocytes and embryos resulted in suppression of mRNA and their protein products, thereby affecting in vitro development of bovine embryos.

scholarly journals Effect of Embryo Density on In Vitro Development and Gene Expression in Bovine In Vitro-fertilized Embryos Cultured in a Microwell System

2013 ◽  
Vol 59 (2) ◽  
pp. 115-122 ◽  
Author(s):  
Satoshi SUGIMURA ◽  
Tomonori AKAI ◽  
Yutaka HASHIYADA ◽  
Yoshio AIKAWA ◽  
Masaki OHTAKE ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Development ◽  
Vitro Development

Effects of hyaluronan, BSA, and serum on bovine embryo in vitro development, ultrastructure, and gene expression patterns

2006 ◽  
Vol 73 (12) ◽  
pp. 1503-1511 ◽  
Author(s):  
A. T. Palasz ◽  
H. Rodriguez-Martinez ◽  
P. Beltran-Breña ◽  
S. Perez-Garnelo ◽  
M. F. Martinez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Patterns ◽  
Gene Expression Patterns ◽  
Bovine Embryo ◽  
In Vitro Development ◽  
Vitro Development
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