279 DIFFERENCES IN TRANSCRIPT ABUNDANCE BETWEEN MALE AND FEMALE BOVINE BLASTOCYSTS PRODUCED IN VITRO USING SEXED SEMEN

There is considerable evidence in the literature indicating that, depending on the culture environment, male pre-implantation embryos develop faster than females, with, for example, a higher proportion of early developing blastocysts being male. The aim of this study was to examine gender-related differences in gene expression in bovine blastocysts produced in vitro following IVF with sex-sorted, frozen–thawed semen. For blastocyst production, immature cumulus–oocyte complexes (COCs) recovered from the ovaries of slaughtered heifers were matured in vitro for 24 h. Matured COCs were randomly split into 2 groups and inseminated with frozen–thawed sperm sorted flow cytometrically for gender. Presumptive zygotes were cultured for 7 days in synthetic oviduct fluid medium. In a preliminary experiment, blastocysts (XX: n = 61; XY: n = 47) were recovered on Day 8 post-insemination and individually snap frozen in liquid nitrogen for verification of the sorting procedure. Sexing was performed with PCR using both male-specific (BRY4.a) and bovine satellite primers. The proportion of female and male blastocysts obtained with X- and Y-chromosome-bearing sperm was 87.2 and 80.3%, respectively. In a subsequent 5 replicates, Day 8 blastocysts were snap frozen in groups of 10 for analysis of mRNA relative abundance. These developmentally important genes were selected based on microarray analysis because they showed a high sensitivity to suboptimal in vitro culture conditions (data not shown). There was no difference in the relative abundance of desmocollin II (DcII), Na/K-ATPase alpha1 subunit (Na/K), ubiquitin-activating enzyme E2 (Ube2), fibroblast growth factor 4 (FGF4), and DNA methyltransferase 1 (Dnmt1) between male and female blastocysts. X-inactive specific transcript (Xist), interferon-tau (IFN), and a Sry-related HMG box transcriptoinal factor (Sox17) were significantly up-regulated in female blastocysts, whereas DNA methyltransferase 3a (Dnmt3a), DNA methyltransferase 3b (Dnmt3b), sarcosine oxidase (Sox), and the transcription factor Oct-4 were significantly up-regulated in males. In conclusion, differences in gene expression between male and female embryos exist and may be related to the well-described differences in the kinetics of development.

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Developmental and molecular correlates of bovine preimplantation embryos

Reproduction ◽

10.1530/rep.1.01021 ◽

2006 ◽

Vol 131 (5) ◽

pp. 895-904 ◽

Cited By ~ 60

Author(s):

Hakan Sagirkaya ◽

Muge Misirlioglu ◽

Abdullah Kaya ◽

Neal L First ◽

John J Parrish ◽

...

Keyword(s):

Gene Expression ◽

Dna Methyltransferase ◽

Expression Profiles ◽

Expression Patterns ◽

Blastocyst Stage ◽

Culture Conditions ◽

Preimplantation Embryos ◽

Developmental Potential

Expression of embryonic genes is altered in different culture conditions, which influence developmental potential both during preimplantation and fetal development. The objective of this study was to define the effects of culture conditions on: bovine embryonic development to blastocyst stage, blastocyst cell number, apoptosis and expression patterns of a panel of developmentally important genes. Bovine embryos were culturedin vitroin three culture media containing amino acids, namely potassium simplex optimization medium (KSOMaa), Charles Rosenkrans 1 (CR1aa) and synthetic oviductal fluid (SOFaa). Apoptosis in blastocysts was determined by TUNEL assay and expression profiles of developmentally important genes were assayed by real-time PCR.In vivo-produced bovine blastocysts were used as controls for experiments determining gene expression patterns. While the cleavage rates did not differ, embryos cultured in SOFaa had higher rates of development to blastocyst stage (P< 0.05). Mean cell numbers and percentages of apoptotic cells per blastocyst did not differ among the groups. Expression of the heat shock protein 70 (Hsp70) gene was significantly up-regulated in both CR1aa and KSOMaa when compared with SOFaa (P< 0.001). DNA methyltransferase 3a (Dnmt3a) expression was higher in embryos cultured in CR1aa than in those cultured in SOFaa (P< 0.001). Expression of interferon tau (IF-τ) and insulin-like growth factor II receptor (Igf-2r) genes was significantly up-regulated in KSOMaa when compared with CR1aa (P< 0.001). Gene expression did not differ betweenin vivo-derived blastocysts and theirin vitro-derived counterparts. In conclusion, SOFaa supports higher development to blastocyst stage than KSOMaa and CR1aa, and the culture conditions influence gene expression.

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243TEMPORAL DIVERGENCE IN THE PATTERN OF MRNA EXPRESSION IN BOVINE EMBRYOS CULTURED FROM THE ZYGOTE TO BLASTOCYST STAGE IN VITRO OR IN VIVO

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab243 ◽

2004 ◽

Vol 16 (2) ◽

pp. 242

Author(s):

P. Lonergan ◽

D. Rizos ◽

A. Gutierrez-Adan ◽

P.M. Moreira ◽

B. Pintado ◽

...

Keyword(s):

Growth Factor ◽

Relative Abundance ◽

Transcript Abundance ◽

Blastocyst Stage ◽

Insulin Like Growth Factor ◽

Bovine Embryos ◽

Interferon Tau ◽

Cell Stage

The objective of this study was to examine the time during the post-fertilization culture period that gene expression patterns of in vitro cultured bovine embryos diverge from those of their in vivo cultured counterparts. Presumptive bovine zygotes were produced by IVM/IVF of immature oocytes collected from the ovaries of slaughtered animals. At approximately 20h post-insemination (hpi), presumptive zygotes were randomly divided into two culture groups, either in vitro in synthetic oviduct fluid or in vivo, and transferred into the ewe oviduct. Embryos were recovered from both systems at approximately 30hpi (2-cell), two (4-cell), three (8-cell), four (16-cell), five (early morula), six (compact morula) or seven (blastocyst) days pi and snap-frozen for the analysis of transcript abundance using real-time PCR. The transcripts studied were interferon-tau, apoptosis regulator box-a (Bax), connexin 43, sarcosine oxidase, glucose transporter 5, mitochondrial Mn-superoxide dismutase, insulin-like growth factor II, and insulin-like growth factor-I receptor, most of which are known from our previous work to be differentially transcribed in blastocysts derived from culture in vitro or in vivo. Analysis was done on pools of 10 embryos. Data were analyzed using one-way repeated measures ANOVA. The relative abundance of the transcripts studied varied throughout the preimplantation period and was strongly influenced by the culture environment. For example, transcripts for interferon-tau were detected from the 8-cell stage onwards in in vitro-cultured embryos but not until the early morula stage in those cultured in vivo. Levels of this transcript increased significantly at the compact morula and blastocyst stages in both groups but were significantly higher (P<0.05) in in vitro-cultured embryos at both stages. mRNA for Bax was not detected before the 8-cell stage in in vitro cultured embryos and not until the 16-cell stage in in vivo cultured embryos. The abundance of this transcript increased significantly thereafter up to the blastocyst stage in both groups. The level of expression was significantly higher (P<0.05) at all stages of development in in vitro-cultured embryos than those cultured in vivo. The relative abundance of Cx43 transcripts decreased in both in vitro- and in vivo-cultured embryos at the 8- to 16-cell stage. Levels remained low thereafter in the in vitro-cultured embryos but significantly increased in those cultured in vivo. Transcript abundance was significantly higher in in vivo cultured embryos from Day 4 onwards with a ten-fold difference presence at the blastocyst stage. Differences also existed for the other transcripts studied. These data demonstrate that changes in transcript abundance in blastocyst stage embryos are in many cases a consequence of perturbed transcription earlier in development. Depending on the transcript, these differences may be evident in as short as 10h of culture.

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148EPIDERMAL GROWTH FACTOR INDUCES BCL-XL GENE EXPRESSION AND REDUCES APOPTOSIS IN PORCINE PARTHENOTES DEVELOPING IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab148 ◽

2004 ◽

Vol 16 (2) ◽

pp. 196

Author(s):

X.S. Cui ◽

Y.J. Jeong ◽

S.H. Cheon ◽

N.-H. Kim

Keyword(s):

Gene Expression ◽

Growth Factor ◽

Relative Abundance ◽

Linear Models ◽

Developmental Rate ◽

Cell Number ◽

Culture Conditions ◽

Preimplantation Embryos ◽

Presence And Absence

Epidermal growth factor (EGF) induces well-documented mitogenic and differentiating effects on murine and bovine preimplantation embryos. However, the effects of EGF on apoptosis and apoptosis-related gene expression in porcine embryos developing in vitro have not been evaluated. The objective of this study was to determine the effects of exogenous EGF in the presence and absence of BSA on the preimplantation development of porcine diploid parthenotes. In addition, we measured cell number, apoptosis, and expression of apoptotic-related genes of the blastocysts that developed in these culture conditions. In vitro-matured oocytes were parthenogenetically activated by square electrical direct current pulses. After 3h of culture in North Carolina State University (NCSU) 23 medium with 0.4% BSA containing 7.5mgmL−1 cytochalasin B, embryos were washed and cultured in NCSU 23 medium with 0.4% BSA for 48h. The general linear models (GLM) procedure in the Statistical Analysis System was used to analyze developmental rates. A paired Student’s t-test was used to compare relative gene expression. Presumptive diploid 4-cell parthenote embryos were randomly cultured in the same medium containing 0 or 10ngmL−1 EGF in the presence and absence of 0.4% BSA. More 4-cell embryos developed into blastocysts at Day 7 when BSA was present at 0.1% (54.8%±4.9) and 0.4% (55.1%±3.1) than when BSA was absent (43.0%±3.5, P<0.05). The addition of 10ngmL−1 EGF into the medium did not significantly increase the developmental rate (40.2±2.2 v. 35.1±2.1), but EGF in the presence of 0.1 and 0.4% BSA significantly increased the cell numbers per blastocyst (51.3±2.8 v. 37.8±2.5 in 0.1% BSA; 51.8±2.0 v. 42.7±2.3 in 0.4% BSA, P<0.01 for both comparisons). Furthermore, EGF treatment in the absence of BSA did not inhibit apoptosis in the blastocysts (6.8%±0.9 v. 8.8%±1.0), while addition of EGF in the presence of 0.4% BSA significantly reduced the degree of apoptosis in the blastocysts (4.0%±0.9 v. 9.3%±1.0, P<0.01). To investigate whether EGF modulates mRNA expression of apoptosis-related genes, mRNA was prepared from single blastocysts and each preparation was subjected to RT-PCR for Bcl-xL and Bak transcripts. EGF enhanced the relative abundance of Bcl-xL expression in the presence of BSA (P<0.01). The relative abundance of Bak expression was not altered by EGF treatment in either the presence or the absence of BSA. These results suggest that EGF and BSA synergistically enhance Bcl-xL gene expression, which may result in a net increase in cell number in porcine presumptive diploid parthenotes developing in vitro.

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Transcriptome comparisons of in vitro intestinal epithelia grown under static and microfluidic gut-on-chip conditions with in vivo human epithelia

Scientific Reports ◽

10.1038/s41598-021-82853-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kornphimol Kulthong ◽

Guido J. E. J. Hooiveld ◽

Loes Duivenvoorde ◽

Ignacio Miro Estruch ◽

Victor Marin ◽

...

Keyword(s):

Gene Expression ◽

Culture Conditions ◽

Gene Set Enrichment Analysis ◽

Shear Stresses ◽

Static Culture ◽

Dynamic Culture ◽

Intestinal Segments ◽

On Chip

AbstractGut-on-chip devices enable exposure of cells to a continuous flow of culture medium, inducing shear stresses and could thus better recapitulate the in vivo human intestinal environment in an in vitro epithelial model compared to static culture methods. We aimed to study if dynamic culture conditions affect the gene expression of Caco-2 cells cultured statically or dynamically in a gut-on-chip device and how these gene expression patterns compared to that of intestinal segments in vivo. For this we applied whole genome transcriptomics. Dynamic culture conditions led to a total of 5927 differentially expressed genes (3280 upregulated and 2647 downregulated genes) compared to static culture conditions. Gene set enrichment analysis revealed upregulated pathways associated with the immune system, signal transduction and cell growth and death, and downregulated pathways associated with drug metabolism, compound digestion and absorption under dynamic culture conditions. Comparison of the in vitro gene expression data with transcriptome profiles of human in vivo duodenum, jejunum, ileum and colon tissue samples showed similarities in gene expression profiles with intestinal segments. It is concluded that both the static and the dynamic gut-on-chip model are suitable to study human intestinal epithelial responses as an alternative for animal models.

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Post-hatching development of bovine embryos in vitro: the effects of tunnel preparation and gender

Zygote ◽

10.1017/s0967199411000086 ◽

2011 ◽

Vol 20 (2) ◽

pp. 123-134 ◽

Cited By ~ 9

Author(s):

Grazieli Marinheiro Machado ◽

Ester Siqueira Caixeta ◽

Carolina Madeira Lucci ◽

Rodolfo Rumpf ◽

Maurício Machaim Franco ◽

...

Keyword(s):

Gene Expression ◽

Morphological Characteristics ◽

Tunnel Construction ◽

Agarose Gel ◽

Male And Female ◽

Embryo Size ◽

And Gender ◽

Phosphate Buffered Saline ◽

Kinetics Of

SummaryThe objective of this study was to compare morphological characteristics, kinetics of development, and gene expression of male and female IVP embryos that were cultured until day (D)15 (fertilization = D0), using either phosphate-buffered saline (PBS) or Milli-Q water (MQW) to dilute the agarose gel used for tunnel construction. On D11, embryos (n = 286) were placed in agarose gel tunnels diluted in PBS and MQW. Embryos were evaluated for morphology, and embryo size was recorded on D11, D12.5, D14 and D15. Then, embryos were sexed and used for gene expression analyses (G6PD, GLUT1, GLUT3, PGK1, PLAC8, KRT8, HSF1 and IFNT). The percentage of elongated embryos at D15 was higher (p < 0.05) in the PBS (54%) than in the MQW (42%) gel. However, embryos produced in MQW were bigger (p < 0.05) and had a lower expression of GLUT1 (p = 0.08) than those cultured in PBS. There was a higher proportion of male than female embryos at D15 in both treatments, MQW (65% vs. 35%; p < 0.05) and PBS (67% vs. 33%; p < 0.05); however, embryo size was not significantly different between genders. Moreover, D15 female embryos had greater expression of G6PD (p = 0.05) and KRT8 (p = 0.03) than male embryos. In conclusion, the diluent used for tunnel construction affected embryo development in the post-hatching development (PHD) system, and the use of MQW was the most indicative measure for the evaluation of embryo quality. Male and female embryos cultured from D11 to D15, either in an MQW or PBS agarose gel, demonstrated similar development but different gene expression.

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Abstract 130: Hypercholesterolemia Suppresses Carotid Artery Endothelial NOS3 Expression via Epigenetic Mechanisms

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.35.suppl_1.130 ◽

2015 ◽

Vol 35 (suppl_1) ◽

Author(s):

Alex Sotolongo ◽

Yi-Zhou Jiang ◽

John Karanian ◽

William Pritchard ◽

Peter Davies

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Endothelial Cells ◽

Dna Methyltransferase ◽

Control Group ◽

High Cholesterol Diet ◽

Cholesterol Diet ◽

High Cholesterol ◽

High Fat

Objective: One of the first clinically detectable changes in the vasculature during atherogenesis is the accumulation of cholesterol within the vessel wall. Hypercholesterolemia is characterized by dysfunctional endothelial-dependent vessel relaxation and impaired NOS3 function. Since DNA methylation at gene promoter regions strongly suppresses gene expression, we postulated that high-fat/high-cholesterol diet suppresses endothelial NOS3 through promoter DNA methylation. Methods: Domestic male pigs were fed control diet (CD) or isocaloric high fat and high cholesterol diet (HC; 12% fat and 1.5% cholesterol) for 2, 4, 8 or 12 weeks prior to tissue collection. Furthermore, to determine the effects of risk factor withdrawal, an additional group of swine received HC for 12 weeks and then CD for 8 weeks; a control group received HC continuously for 20 weeks. Endothelial cells were harvested from common carotid aorta. In parallel in vitro studies, cultured human aortic endothelial cells (HAEC) were treated with human LDL, GW3956 (LXR agonist) and RG108 (DNA methyltransferase [DNMT] inhibitor). In cells from both sources, DNA methylation at the NOS3 promoter was measured using methylation specific pyro sequencing, and endothelial gene expression was measured using RT PCR. Results: HC diet increased plasma cholesterol level from 75 mg/dl on CD to a plateau of about 540 mg/dl within 2 weeks. Endothelial NOS3 expression was significantly reduced (71±9 % of CD) after 4 weeks of HC, a level sustained at subsequent time points. Withdrawal of HC for 8 weeks did not recover NOS3 expression. After 12-week HC, the NOS3 promoter was hypermethylated. Withdrawal of HC did not reverse NOS3 promoter methylation. In vitro treatment of HAEC with human LDL (200 mg/dl total cholesterol) or GW3956 (5μM) suppressed NOS3 mRNA to 50% and 30% respectively, suggesting that LXR/RXR is involved in suppression of NOS3. Nitric oxide production was consistently suppressed by GW3959. Both could be reversed through inhibition of DNMTs by RG108. Conclusions: DNA methylation and LXR/RXR pathway can mediate the HC-suppression of endothelial NOS3. The study identifies novel pharmaceutical targets in treating endothelial dysfunction. Crosstalk between these pathways is under investigation.

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Genetic determinants of growth phase-dependent and adenovirus 5-responsive expression of the Chinese hamster thymidine kinase gene are contained within thymidine kinase mRNA sequences.

Molecular and Cellular Biology ◽

10.1128/mcb.6.6.2262 ◽

1986 ◽

Vol 6 (6) ◽

pp. 2262-2266 ◽

Cited By ~ 12

Author(s):

J A Lewis ◽

D A Matkovich

Keyword(s):

Gene Expression ◽

Cell Culture ◽

Thymidine Kinase ◽

Growth Phase ◽

Chinese Hamster ◽

Culture Conditions ◽

Kinase Gene ◽

Simplex Virus ◽

Adenovirus 5

We have constructed a chimeric thymidine kinase (TK) minigene, pHe delta 6Ha, which combines the complete coding and 3' noncoding regions of a Chinese hamster TK cDNA with the promoter region and 5' untranslated region of the TK gene of herpes simplex virus type 1. We have transformed rat 4 cells to Tk+ with this gene and analyzed the pattern of TK gene expression in these transformants under various conditions of in vitro cell culture. We find that TK gene expression in these Tk+ transformants is growth phase dependent, responsive to adenovirus 5 infection, and indistinguishable in character under a variety of cell culture conditions from the pattern of TK gene expression in rat 4 cells transformed to Tk+ with the genomic Chinese hamster TK gene clone lambda HaTK.5. We are led to the conclusion that the genetic elements which mediate growth phase-dependent TK gene expression are contained entirely within the sequences of the mature cytoplasmic hamster TK mRNA.

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Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab136 ◽

2021 ◽

Author(s):

Yu Takahashi ◽

Yu Inoue ◽

Keitaro Kuze ◽

Shintaro Sato ◽

Makoto Shimizu ◽

...

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Gene Expression Regulation ◽

Intestinal Epithelial Cells ◽

Three Dimensional ◽

Culture Conditions ◽

Dependent Manner ◽

Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

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193 DIFFERENTIAL EFFECTS OF CULTURE ON APOPTOTIC GENE EXPRESSION IN THE PRE-IMPLANTATION CLONED EMBRYOS OF MINIATURE PIG

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab193 ◽

2007 ◽

Vol 19 (1) ◽

pp. 213

Author(s):

M. R. Park ◽

I. S. Hwang ◽

H. J. Moon ◽

J. H. Shim ◽

D. H. Kim ◽

...

Keyword(s):

Gene Expression ◽

Relative Abundance ◽

Total Concentration ◽

Control Group ◽

Specific Cell ◽

Miniature Pig ◽

Cell Stage ◽

Apoptotic Gene ◽

Early Embryos

Manipulations of early embryos require that the embryos be placed in vitro. The ability to reproduce in vivo conditions in vitro would greatly facilitate studies on the development of early embryos. A variety of different conditions have been described that result in development of pig embryos from the 1-cell stage to the blastocyst stage in vitro. There is a species-specific cell stage at which the early embryo is very sensitive to in vitro conditions, which generally corresponds to the stage at which the embryo begins producing significant amounts of RNA. The present study was conducted to investigate the relative amounts of apoptotic gene expression in miniature pig NT embryos under culture conditions of different osmolarity. Oocytes were cultured in TCM-199 for 40–44 h at 38.5�C under 5% CO2 in air. Miniature pig ear fibroblast cells were cultured to reach confluency, and the culture was continued for an additional 5–6 days. The NaCl group of embryos was cultured in PZM-3 supplemented with 138 mM NaCl in total concentration (280–320 mOsmol) for the first 2 days, and then cultured in PZM-3 (250–270 mOsmol) for a further 4 days. The control group of embryos was cultured in the PZM-3 for the entire period of in vitro culture. Total RNA samples were prepared from 2 blastocysts using the Roche 1st strand cDNA synthesis kit. Bax and Bcl-xl gene expression of blastocysts was analyzed by real-time RT-PCR. Developemntal rates were analyzed by a GLM procedure of SAS (SAS Institute, Inc., Cary, NC, USA). Relative gene expression was compared by Student's t-test. Blastocyst formation rate in the NaCl group was not different from that in the control group (25.4% and 23.2%, respectively), but the apoptosis rate was significantly lower (P < 0.05) in the NaCl group (1.6%) than in the control (7.1%). The relative abundance of Bax mRNA expression was significantly higher (P < 0.05) in the control group (n = 32) than in the NaCl group (n = 33). However, the relative abundance of Bcl-xl mRNA was significantly higher (P < 0.05) in NaCl group. The relative abundance of Bax/Bcl-xl was significantly higher in the control group than in the NaCl group (P < 0.05). These results indicate that the hypertonic culture condition at the early embryonic stage of miniature pig NT embryos could reduce the frequency of apoptosis through regulating Bax and Bcl-xl gene expression.

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187 INCREASED NUMBER OF EXPANDED BOVINE BLASTOCYSTS IN SOF AND CR1 RELATIVE TO KSOM

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab187 ◽

2007 ◽

Vol 19 (1) ◽

pp. 210

Author(s):

D. M. Kohl ◽

R. L. Monson ◽

L. E. Enwall ◽

J. J. Rutledge

Keyword(s):

Gene Expression ◽

Culture Media ◽

Expression Profiles ◽

Expression Patterns ◽

Culture Conditions ◽

Pregnancy Rates ◽

Embryo Morphology ◽

Bovine Embryos ◽

Chi Square Analysis

Assessment of morphological stage grade is a subjective procedure. Stage grade is of vital importance to, among other things, recipient synchrony for the purpose of establishing successful pregnancies. Asynchronous embryo transfer has led to decreases in pregnancy rates (Farin et al. 1995 Biol. Reprod. 52, 676–682) and has been implicated in contributing to large offspring syndrome (Young et al. 1996 Theriogenology 45, 231). Differences in embryo kinetics based on culture conditions have been well documented (Mello et al. 2005 Reprod. Fert. Dev. 17, 221 abst). Whether such differences are the result of species, breed, metabolic stress, sire effects, or separation from an in vivo environment has yet to be determined. The correlation between oxygen respiration rates and embryo morphology as well as embryo diameter in bovine embryos produced in vitro has shown promise in the development of a more objective predictor of embryo quality and perhaps pregnancy initiation (Lopes et al. 2005 Reprod. Fert. Dev. 17, 151 abst). As well, recent examination of gene expression patterns of in vitro-derived bovine embryos seems to indicate that longer periods of in vitro culture are associated with lower rates of embryo survival (Lonergan et al. 2006 Theriogenology 65, 137–152). We hypothesize that differences do exist in the number, rate, and morphological appearance of blastocysts and that these parameters are in large part based on culture conditions in vitro. The objective of this experiment was to determine the timing and distribution of blastocyst formation of in vitro-produced bovine embryos cultured in SOF8, CR18AA, and KSOM8, under a standard incubation environment. Bovine ovaries from a local abattoir were aspirated and matured for 18-22. Oocytes were fertilized with frozen-thawed Percoll-separated semen from a Holstein bull. Presumptive zygotes were vortexed to remove cumulus cells and placed into 3 different culture media in a highly humidified atmosphere containing 20% oxygen, 5% carbon dioxide, and compressed air at 38.5�C. Embryos were evaluated specifically at 168 h post-insemination (Day 7) and assigned a morphological stage grade (IETS) to determine fixed time point differences. A total of 6 complete replicates were performed. Only embryos exhibiting the presence of a blastocoel at this time were documented (early blast, mid-blast, expanded blast). At 168 h post-insemination, there were no significant differences in the total number of embryos reaching early or mid-blast stage in any of the media. However, chi-square analysis revealed an increase in the number of expanded blastocysts in SOF (n = 813) and CR1 (n = 838) treatments compared to KSOM (n = 824; P < 0.0001). Expanded blastocysts in SOF were also greater in number than in CR1 (P < 0.05). Embryo selection based on development to the expanded blastocyst stage on Day 7 may prove useful in increasing pregnancy rates, and may validate qualitative correlations based on oxygen consumption and gene expression profiles for embryos produced in vitro.

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