35 EFFECT OF CULTURE AT LOW OR ATMOSPHERIC OXYGEN TENSION IN SOMATIC DONOR CELLS FOR HORSE NUCLEAR TRANSFER

2013 ◽  
Vol 25 (1) ◽  
pp. 165
Author(s):  
A. Gambini ◽  
J. Jarazo ◽  
A. De Stefano ◽  
F. Karlaninan ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Oxygen Tension ◽  
Embryo Development ◽  
Atmospheric Oxygen ◽  
Culture Conditions ◽  
Chi Square ◽  
Reconstructed Embryos ◽  
Donor Cells

Somatic donor cells play a major role during the NT procedure. In vitro culture conditions may affect the capability of these cells to be reprogrammed and to allow embryo development. The aim of this study was to evaluate the effect of in vitro culture at low (5%) or atmospheric (20%) oxygen tension in somatic donor cells for cloned equine embryo production. Adult fibroblasts were obtained through culture of minced tissue from neck biopsies of one horse skin. They were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics in 2 groups: (1) 5% CO2 and (2) 5% CO2 and 5% O2, both groups in humidified air at 39°C. Quiescence of donor cells was induced by growth to confluency for 3 to 5 days prior to NT. Oocyte collection, maturation, cloning, and activation procedures were performed as described by Gambini et al. (2012 Biol. Reprod. 87, 1–9.). After activation, reconstructed embryos (RE) were cultured in DMEM/F12 supplemented with 5% FBS in the well of the well system as 3 reconstructed embryos per well. Cleavage and blastocyst formation (7–8 days) of the experimental groups were assessed. In vitro development, on a per-well and RE basis, was compared using the chi-square test. No statistical differences were observed in cleavage [(1): 48/84, 57%; (2): 54/87, 62%). No difference was observed in blastocyst rates on a per-well basis [(1): 5/28, 18%; (2): 4/29, 14%] or on a per-RE basis [(1): 5/84, 6%; (2): 4/87, 5%]. This work suggests that the oxygen tension during the in vitro culture of somatic donor cells does not affect the quantity of the cloned equine blastocyst produced. Further studies are required to determine if these conditions would affect in vivo embryo development.

Embryo culture at a reduced oxygen concentration of 5%: a mini review

Zygote ◽  
2019 ◽  
Vol 27 (6) ◽  
pp. 355-361 ◽  
Author(s):  
R. Sciorio ◽  
G.D. Smith
Keyword(s):  
Oxygen Tension ◽  
Embryo Development ◽  
Atmospheric Oxygen ◽  
Embryo Implantation ◽  
Critical Role ◽  
Low Oxygen ◽  
Physiological Level ◽  
Low Oxygen Tension

SummaryThe optimum oxygen tension for culturing mammalian embryos has been widely debated by the scientific community. While several laboratories have moved to using 5% as the value for oxygen tension, the majority of modern in vitro fertilization (IVF) laboratory programmes still use 20%. Several in vivo studies have shown the oxygen tension measured in the oviduct of mammals fluctuates between 2% and 8% and in cows and primates this values drops to <2% in the uterine milieu. In human IVF, a non-physiological level of 20% oxygen has been used in the past. However, several studies have shown that atmospheric oxygen introduces adverse effects to embryo development, not limited to numerous molecular and cellular physiology events. In addition, low oxygen tension plays a critical role in reducing the high level of detrimental reactive oxygen species within cells, influences embryonic gene expression, helps with embryo metabolism of glucose, and enhances embryo development to the blastocyst stage. Collectively, this improves embryo implantation potential. However, clinical studies have yielded contradictory results. In almost all reports, some level of improvement has been identified in embryo development or implantation, without any observed drawbacks. This review article will examine the recent literature and discusses ongoing efforts to understand the benefits that low oxygen tension can bring to mammal embryo development in vitro.

scholarly journals Effect of insulin-like growth factor-1 during in vitro oocyte maturation and in vitro culture of bovine embryos

2001 ◽  
Vol 53 (2) ◽  
pp. 207-211 ◽  
Author(s):  
M.D. Quetglas ◽  
L.A. Coelho ◽  
J.M. Garcia ◽  
E.B. Oliveira Filho ◽  
C.R. Esper
Keyword(s):  
Growth Factor ◽  
In Vitro Culture ◽  
Embryo Development ◽  
Calf Serum ◽  
Culture Conditions ◽  
Insulin Like Growth Factor ◽  
Chi Square ◽  
Igf I ◽  
Chi Square Test

The effects of insulin-like growth factor-I (IGF-I) on in vitro maturation (IVM) (experiment I) and on in vitro embryo development (experiment II) of bovine oocytes fertilized in vitro, were evaluated in terms of cleavage (CR), blastocyst (BR) and hatching (HR) rates. For IVM, immature cumulus-oocyte complexes were cultured in TCM-199 medium supplemented with Hepes, sodium bicarbonate, sodium pyruvate, additives, fetal calf serum (B-199 medium) and gonadotropins (14 U/ml PMSG and 7 U/ml hCG). For embryo development, the oocytes/zygotes were cultured in B-199 medium with bovine oviduct epithelial cells in suspension under silicon oil. Treatments for in vitro culture conditions for both experiments were: 1- B-199 + 200 ng/ml IGF-I; 2- B-199 + 100 ng/ml IGF-I; 3- B-199 + 50 ng/ml IGF-I; 4- B-199 + 10 ng/ml IGF-I; 5- B-199 + 0 ng/ml IGF-I. All cultures were performed at 38.5ºC in 5% CO2 in air and the data were analyzed by chi-square test. In experiment I, there were no differences (P>0.05) among treatments for CR, BR or HR. In experiment II, the addition of IGF-I to the embryo culture medium (ECM) resulted in a significant increase in CR while for BR and HR this effect was not observed. The addition of 200 ng/ml IGF-I to ECM increased CR (71.1%) when compared to 100 ng/ml IGF-I (57.6%) or control (56.7%) groups, however, there were no differences when compared to 50 (69.4%) or 10 ng/ml (73.1%) groups. There was no beneficial effect of the addition of IGF-I in the IVM or ECM media on the in vitro development of embryos produced from oocytes matured and fertilized in vitro.

scholarly journals The Role of Tissue Oxygen Tension in Dengue Virus Replication

Cells ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 241 ◽  
Author(s):  
Efseveia Frakolaki ◽  
Panagiota Kaimou ◽  
Maria Moraiti ◽  
Katerina Kalliampakou ◽  
Kalliopi Karampetsou ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Oxygen Tension ◽  
Atmospheric Oxygen ◽  
Rna Replication ◽  
Metabolic Reprogramming ◽  
Anaerobic Glycolysis ◽  
Low Oxygen ◽  
Hypoxia Response

Low oxygen tension exerts a profound effect on the replication of several DNA and RNA viruses. In vitro propagation of Dengue virus (DENV) has been conventionally studied under atmospheric oxygen levels despite that in vivo, the tissue microenvironment is hypoxic. Here, we compared the efficiency of DENV replication in liver cells, monocytes, and epithelial cells under hypoxic and normoxic conditions, investigated the ability of DENV to induce a hypoxia response and metabolic reprogramming and determined the underlying molecular mechanism. In DENV-infected cells, hypoxia had no effect on virus entry and RNA translation, but enhanced RNA replication. Overexpression and silencing approaches as well as chemical inhibition and energy substrate exchanging experiments showed that hypoxia-mediated enhancement of DENV replication depends on the activation of the key metabolic regulators hypoxia-inducible factors 1α/2α (HIF-1α/2α) and the serine/threonine kinase AKT. Enhanced RNA replication correlates directly with an increase in anaerobic glycolysis producing elevated ATP levels. Additionally, DENV activates HIF and anaerobic glycolysis markers. Finally, reactive oxygen species were shown to contribute, at least in part through HIF, both to the hypoxia-mediated increase of DENV replication and to virus-induced hypoxic reprogramming. These suggest that DENV manipulates hypoxia response and oxygen-dependent metabolic reprogramming for efficient viral replication.

129 DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES AFTER IN VITRO MATURATION AND IN VITRO CULTURE UNDER COMPARATIVE OXYGEN TENSION

2011 ◽  
Vol 23 (1) ◽  
pp. 169
Author(s):  
J. T. Kang ◽  
M. Atikuzzaman ◽  
D. K. Kwon ◽  
S. J. Park ◽  
S. J. Kim ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Oxygen Tension ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Mrna Abundance ◽  
Developmental Competence ◽  
Multiple Genes ◽  
Oxygen Concentrations

The in vitro developmental abilities of porcine oocytes are generally increasing steadily at a similar ratio to those of in vivo embryos. However, it has been suggested that the in vitro culture system for the development of porcine embryos is not optimal. In this study, we investigated the effect of 2 oxygen concentrations (5 and 20%) on porcine embryo development during in vitro maturation and in vitro culture and analyzed differences in gene expression of resulting blastocysts. Oocytes were recovered by aspiration of slaughterhouse ovaries and then matured in tissue culture medium (TCM) 199 supplemented with 10% porcine follicular fluid (pFF), epidermal growth factor (EGF), insulin, pyruvate, cystine, and gonadotropin. Matured oocytes were then activated parthenogenetically, cultured in PZM-3 media for 7 days. In vitro maturation (M group) of oocytes was carried out under two oxygen concentration (5 and 20%) in terms of nuclear maturation (polar body extrusion; Exp. 1). The developmental differences between 5% oxygen culture group and 20% oxygen culture group during in vitro culture (C group) of embryos after parthenogenetic activation was investigated in terms of first cleavage and blastocyst formation (Exp. 2). Relative mRNA abundance of multiple genes in blastocysts was analyzed for transcript abundance of genes related with metabolism (GLUT1, LDHA), oxidative response (MnSOD, GPX1), apoptosis (BAX, Bcl2), and developmental competence (CCNB1, IGF2R; Exp. 3). The results show there were no significant differences in maturation rate between 2 oxygen concentrations during in vitro maturation (83 v. 86%). It was thought that cumulus cells surrounding oocytes might have attenuated oxidative stress, but number of resulting blastocysts were (P < 0.05) increased in 5% IVC group when compared with 20% IVC group (18.67 v. 14.09%, respectively). Moreover, the M20C5 group (23.01%) had a beneficial effect on in vitro culture compared with M5C5 (14.32%), M5C20 (10.30%), and M20C20 (17.88%) groups. Total cell numbers were not significantly different among groups. According to mRNA abundance data of multiple genes, each group altered the expression of genes in various patterns. Therefore, it could be concluded that high oxygen tension during in vitro maturation and low oxygen tension during in vitro culture might alter the expression of multiple genes related to oocyte competence and improve (P < 0.05) embryo development, but not blastocyst quality. This study was supported by MKE (#2009-67-10033839, #2009-67-10033805), NRF (#M10625030005-508-10N25), BK21 for Veterinary Science, IPET (#109023-05-1-CG000), and Hanhwa L&C.

35 EFFECTIVENESS OF MICROWELL-BASED IN VITRO CULTURE SYSTEMS FOR BOVINEZONA-FREE CLONED EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 124
Author(s):  
C. Feltrin ◽  
M. Machado ◽  
L. M. V. Queiroz ◽  
M. A. S. Peixer ◽  
P. F. Malard ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Aggregation State ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Vitro Development

In vitro embryo production by handmade cloning (HMC) usually requires individual embryo culture, because zona-free embryos cannot be grouped in standard in vitro culture (IVC) protocols. The aim of this study was to evaluate the developmental potential of bovine embryos produced by HMC (Ribeiro et al. 2009 Cloning Stem Cells 11, 377–386) after in vitro culture (IVC) in 3 microwell (WOW) systems. After in vitro maturation, oocytes were denuded and incubated in demecolcine (Ibáñez et al. 2003 Biol. Reprod. 68, 1249–1258), followed by zona pellucida removal, oocyte bisection, embryo reconstruction, electrofusion, and chemical activation. Cloned embryos were allocated to 1 of 3 IVC groups: cWOW: conventional microwells (250 μm, round; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264); mWOW: modified microwells (130 μm, conical; Feltrin et al. 2006 Reprod. Fert. Dev. 18, 126); and WOW-PDMS: microwells in polydimethylsiloxane chips (170 μm, cylindrical with microchannels); IVF embryos were used as controls (Bertolini et al. 2004 Reproduction 128, 341–354). Cleavage (Day 2), blastocyst (Day 7), and pregnancy (Day 30) rates were analysed by the chi-square test, for P < 0.05. Results are shown in Table 1. Cleavage rates were similar between groups, but development to the blastocyst stage was higher in IVF controls than cloned embryo groups. Among cloned embryo groups, blastocyst rate was higher in the mWOW group than the conventional and the PMDS-based microchannels. Nevertheless, in vivo development to Day 30 of pregnancy was not different between cloned groups. Our results for in vitro embryo development indicated that the mWOW provided more suitable conditions for embryo development to the blastocyst stage when compared with cWOW or even WOW-PDMS. Among some possible reasons include the physical advantage of a smaller microwell that may better mimic the constraining effect of the zona pellucida on the developing embryo. That may also provide greater blastomere stability, favouring the aggregation state during the first rounds of cleavages, also aiding compaction and subsequent cavitation. The narrower microwell system appeared to have promoted better in vitro development than the conventional and the DMPS-based microwell systems, with no impact on subsequent in vivo development. However, the IVC in the WOW-PDMS system supported reasonable rates of development, in accordance with the current literature. Table 1.In vitro development of bovine IVF and cloned embryos produced after the in vitro culture in distinct IVC systems

An ultrastructural study of embryo and endosperm development during in vitro culture of maize ovaries (Zea mays)

1986 ◽  
Vol 64 (10) ◽  
pp. 2227-2238 ◽  
Author(s):  
J. H. N. Schel ◽  
H. Kieft
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Light And Electron Microscopy ◽  
Culture Method ◽  
Endosperm Development ◽  
Endosperm Formation ◽  
Development In Vitro ◽  
Maize Embryos

A culture method is described which allows the continuous supply of fresh liquid medium and which prevents the accumulation of toxic metabolites. Development of maize embryos and endosperm after various periods of in vitro ovary culture was studied by light and electron microscopy. Using this method the ultrastructural features of embryo development in vitro were similar to those of in vivo embryos. In contrast, the formation of endosperm was irregular with the absence of cellularization of the inner endosperm being frequent. In some cases, only the endosperm developed without any indication of embryo formation. In a calcium-depleted medium, embryo development was normal but again, endosperm formation was aberrant. No cells were formed in the central part of the endosperm and near the placental region degeneration took place, resulting in vacuoles with dark inclusions, clumps of rough endoplasmic reticulum membranes, and cellular breakdown. The events occurring after in vitro culture strongly resemble those taking place after intergeneric crosses or crosses between diploid and tetraploid strains. It is concluded that defective endosperm development is probably the main factor for the failure of embryo development.

27 SOMATIC CELL NUCLEAR TRANSFER CLONING AND EMBRYO AGGREGATION IN PIGS

2014 ◽  
Vol 26 (1) ◽  
pp. 128
Author(s):  
C. P. Buemo ◽  
A. Gambini ◽  
I. Hiriart ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Electric Pulse ◽  
Blastocyst Stage ◽  
Control Group

Somatic cell nuclear transfer (SCNT) derived blastocysts have lower cell number than IVF-derived blastocysts and their in vivo counterparts. The aim of this study was to improve the blastocyst rates and quality of SCNT blastocysts by the aggregation of genetically identical free zona pellucida (ZP) porcine clones. Cumulus–oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the ZP using a protease and then enucleated by micromanipulation; staining was performed with Hoechst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80 V for 30 μs). All reconstituted embryos (RE) were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% PVA) by a DC pulse of 1.2 kV cm–1 for 80 μs. Then, the oocytes were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of free ZP embryos was achieved in a system of well of wells in 100 μL of medium, placing 3 activated oocytes per microwell (aggregation embryo), whereas the control group was cultivated with equal drops without microwells. Embryos were cultivated at 39°C in 5% O2, 5% CO2 for 7 days in SOF medium with a supplement of 10% fetal bovine serum on the fifth day. The RE were placed in microwells. Two experimental groups were used, control group (not added 1X) and 3 RE per microwell (3X). At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality and evaluate if the embryo aggregation improves it. Results demonstrated that aggregation improves in vitro embryo development rates until blastocyst stage and indicated that blastocysts rates calculated over total number of oocytes do not differ between groups (Table 1). Embryo aggregation improves cleavage per oocyte and cleavage per microwell rates, presenting statistical significant differences and increasing the probabilities of higher embryo development generation until the blastocyst stage with better quality and higher diameter. Table 1.Somatic cell nuclear transfer cloning and embryo aggregation

211 IN VITRO CULTURE CONDITIONS AFFECT GENE EXPRESSION PATTERN OF BOVINE BLASTOCYST IN A STAGE-SPECIFIC MANNER

2013 ◽  
Vol 25 (1) ◽  
pp. 254 ◽  
Author(s):  
A. Gad ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Hölker ◽  
M. U. Cinar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
In Vitro Culture ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Control Group ◽  
Transcriptome Profile ◽  
Specific Manner

The aim of this study was to examine the effect of in vitro culture conditions at specific phases of early embryonic development on the transcriptome profile of bovine blastocysts. Simmental heifers were superovulated and artificially inseminated 2 times with the same frozen–thawed commercial bull semen. Using nonsurgical endoscopic oviductal flushing technology (Besenfelder et al. 2001 Theriogenology 55, 837–845), 6 different blastocyst groups were flushed out at different time points (2-, 4-, 8-, 16-, 32-cell and morula). After flushing, embryos cultured under in vitro conditions until the blastocyst stage. Blastocysts from each group were collected and pooled in groups of 10. Complete in vivo blastocysts were produced and used as control. A unique custom microarray (Agilent) containing 42 242 oligo probes (60-mers) was used over 6 replicates of each group v. the in vivo control group to examine the transcriptome profile of blastocysts. A clear difference in terms of the number of differentially expressed genes (DEG, fold change ≥2, false discovery rate ≤0.05) has been found between groups flushed out at 2-, 4-, and 8-cell (1714, 1918, 1292 DEG, respectively) and those flushed out at 16-, 32-cell and morula stages and cultured in vitro until blastocyst stage (311, 437, 773 DEG, respectively) compared with the complete vivo group. Ontological classification of DEG showed cell death to be the most significant function in all groups. However, the longer time embryos spent under in vitro conditions, the more the percentage of DEG involved in cell death and apoptosis processes are represented in those groups. In addition, genes related to post-translational modification and gene expression processes were significantly dysregulated in all groups. Pathway analysis revealed that protein ubiquitination pathway was the dominant pathway in the groups flushed out at 2-, 4-, and 8-cells but not in the other groups flushed at later stages compared with the in vivo control group. Moreover, retinoic acid receptor activation and apoptosis signalling pathways followed the same pattern. Embryos flushed out before the time of embryonic genome activation and subsequently cultured in vitro were highly affected by culture conditions. Overall, the results of the present study showed that despite the fact that embryos originated from the same source, in vitro culture condition affected embryo quality, measured in terms of gene expression, in a stage-specific manner.

Capacitation and the acrosome reaction in marsupial spermatozoa

1996 ◽  
Vol 8 (4) ◽  
pp. 595 ◽  
Author(s):  
KE Mate ◽  
JC Rodger
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Acrosome Reaction ◽  
Culture Conditions ◽  
Current Understanding ◽  
Critical Event ◽  
Vitro Fertilization ◽  
Australian Species

Although yet to be established definitively, it appears that marsupial spermatozoa require a process of capacitation and that the mechanisms involved may be quite different between the Australian and American species. For Australian species, failure to induce this functional event in culture has meant that in vitro fertilization (IVF) is yet to be achieved. However, in the American species with paired spermatozoa, IVF and subsequent embryo development have been obtained under quite simple culture conditions. Our understanding of the interactions of marsupial spermatozoa with the female tract, and in particular the oviduct, the most likely site of capacitation, is discussed. Although the acrosome reaction (AR) is an equally critical event in marsupial fertilization it appears to be regulated quite differently. The uniquely stable character of the marsupial acrosome is examined as well as our current understanding of the regulation of the marsupial sperm AR in vivo and in vitro.

134 TRANSCRIPTOME PROFILE OF BOVINE BLASTOCYSTS DERIVED FROM ALTERNATIVE IN VIVO AND IN VITRO CULTURE CONDITIONS AT SPECIFIC PHASES OF EARLY EMBRYONIC DEVELOPMENT

2012 ◽  
Vol 24 (1) ◽  
pp. 179 ◽  
Author(s):  
A. Gad ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Hölker ◽  
M. U. Cinar ◽  
...  
Keyword(s):  
Embryonic Development ◽  
In Vitro Culture ◽  
In Vitro Maturation ◽  
Expression Patterns ◽  
Culture Conditions ◽  
Control Group ◽  
Transcriptome Profile ◽  
Vitro Fertilization

An understanding of gene expression patterns due to altered environmental conditions during different time points of the pre-implantation period would improve our knowledge on regulation of embryonic development and improve success of embryo culture. The aim of this study was to examine the effect of alternative in vivo and in vitro culture conditions at specific phases of early embryonic development on transcriptome profile of bovine blastocysts. Using nonsurgical endoscopic oviducal transfer technology, 5 different blastocyst groups were produced. The first 2 groups were matured in vitro and then either transferred after maturation or after in vitro fertilization to synchronized recipients. The other 3 groups were matured, fertilized and cultured in vitro until 4-cell, 16-cell and morula stage before transfer. Blastocysts from each group were collected by uterine flushing at Day 7 and pooled in groups of 10. Complete in vitro (IVP) and in vivo blastocysts were produced and used as controls. A unique custom microarray (Agilent) containing 42 242 oligo probes (60-mers) was used over 6 replicates of each group vs the in vivo control group to examine the transcriptome profile of blastocysts. Compared with the in vivo control group, clear dramatic shifts were found in the number of differentially expressed genes (DEG, fold change ≥2) at 2 different time points. The first shift occurred for blastocyst groups that were transferred after in vitro fertilization and before embryonic genome activation (EGA). The second shift occurred for blastocyst groups that were transferred after EGA, as well as for the IVP group. Ontological classification of DEG showed that the more time spent under in vitro conditions, the higher the percentage of DEG involved in cell death and apoptotic processes. Moreover, lipid metabolism was the most significant process affected in the blastocysts transferred after in vitro maturation and blastocysts transferred at 16-cell stage. Most DEG involved in this process were down-regulated. Pathway analysis revealed that signalling pathways were the dominant pathways in all groups except the group that was transferred after in vitro maturation. That group showed significant down-regulation for genes involved in retinoic acid receptors activation pathways. These results showed that fertilization and EGA were the most critical developmental stages influenced by in vitro culture conditions and subsequently affect blastocyst quality, as measured in terms of gene expression patterns. Moreover, we identified molecular mechanisms and pathways that were influenced by altered culture conditions. These findings will enable the examination of strategies for modifying in vitro culture conditions at critical stages that will allow more efficient production of developmentally competent blastocysts.

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