94 EFFICIENT BOVINE EMBRYO SPLITTING FOR GENE EXPRESSION AND IN VIVO DEVELOPMENT STUDIES

2014 ◽  
Vol 26 (1) ◽  
pp. 161
Author(s):  
A. Velasquez ◽  
D. Veraguas ◽  
F. O. Castro ◽  
J. F. Cox ◽  
L. l. Rodriguez-Alvarez
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Gene Expression Pattern ◽  
Blastocyst Stage ◽  
Embryo Morphology ◽  
Bovine Embryo ◽  
Gene Markers ◽  
Developmental Potential

It is known that embryos produced in vitro are less competent than their in vivo-derived counterparts. When embryos are produced or manipulated in vitro, their developmental potential decreases significantly, which impinges upon the production of viable offspring. In bovines, embryos that will be transferred to a surrogate mother are selected at the blastocysts stage using noninvasive methods, such as their morphological features. However, many of those embryos are not able to implant or to maintain a normal pregnancy because embryo morphology does not reflect its developmental potential and a correct gene expression pattern that support a normal development. It seems that the ideal method for embryo selection would be based on the screening of gene markers that correlate with successful pregnancy after embryo transfer. In that sense, we have proposed an approach to characterise gene expression pattern of early (Day 7) bovine blastocysts and to correlate this gene expression with further developmental potential in vivo, i.e. upon elongation until Day 17. For that, it was established an efficient method to produce identical and viable hemi-embryos by splitting IVF bovine blastocysts in order to set the expression profile of certain genes in one hemi-embryo at blastocyst stage, while the counterpart embryo elongates in vivo for 10 days. A total of 129 blastocysts were split. Six groups of blastocysts were used for splitting and the results compared: 1) Day-7 early blastocysts (n = 20); 2) Day-7 expanded blastocysts (n = 25); 3) Day-7 hatched blastocysts (n = 17); 4) Day-8 early blastocysts (n = 10); 5) Day-8 expanded blastocysts (n = 12); and 6) Day-8 hatched blastocysts (n = 45). Hemi-embryos derived from day-8 grade I and well expanded blastocysts had the greatest survival rate, in vitro re-expansion (67.7%; P < 0.05) and both hemi-embryos conserved a normal morphology with a total cell number over 80 after 6 h in culture. Also both hemi-embryos at blastocyst stage showed homogeneous expression pattern of the genes OCT4, SOX2, NANOG, CDX2, ACTB, and GAPDH (P < 0.05). Finally, the in vivo survival of hemi-embryos was assessed and compared with nonsplit embryos (control) by transferring to recipient cow and collecting at Day 17 of development. For this, hemi-embryos derived from Day-8 hatched blastocyst were used. From 14 transferred hemi-embryos, 5 (35.7%) were collected, and 9 elongated from 17 controls were recovered (52.9%). Also the elongation rate was significantly lower in hemi-embryos than in control; the length of hemi-embryos had a range between 1 and 5 cm, whereas 60% of the control embryos were longer than 10 cm. Our results provide an initial approach to study the correlation among the gene expression characteristics of early bovine embryos with their further development. However, it seems that embryo splitting hampers their elongation in vivo. It might be possible that the development of split embryos is retarded because of manipulation. This work was partially supported by Fondecyt grant no. 11100082 from the Ministry of Education of Chile.

57 GENE EXPRESSION PATTERN OF MINIATURE PIG SOMATIC CELL NUCLEAR TRANSFER EMBRYOS TREATED WITH THE HISTONE DEACETYLASE INHIBITOR SCRIPTAID

2011 ◽  
Vol 23 (1) ◽  
pp. 134
Author(s):  
C. H. Park ◽  
S. G. Lee ◽  
H. J. Lee ◽  
T. K. Jung ◽  
Y. H. Jeong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Histone Deacetylase ◽  
Expression Pattern ◽  
Histone Deacetylase Inhibitor ◽  
Gene Expression Pattern ◽  
Control Group ◽  
Developmental Potential ◽  
Deacetylase Inhibitor

It was recently shown that treatment of cloned embryos with histone deacetylase inhibitors improves efficiency for the success rate of developmental potential to term in several species. The objective of the present study was to investigate the influence of the histone deacetylase inhibitor Scriptaid (Sc) on in vitro development in early porcine SCNT embryos and on their gene expression pattern. Based on the findings of previous porcine studies (Zhao et al. 2009), the reconstructed oocytes were treated with 500 nM Scriptaid for 14 to 16 h after post-fusion activation (6-DMAP/demecolcine). In our preliminary study, blastocyst rate significantly increased in the Sc-treated group, compared with the control group (25.1 ± 2.8% and 13.8 ± 1.9%, respectively, P < 0.05). We determined gene expression using quantitative real-time RT-PCR. The results showed that OCT3/4 gene was expressed at a similar level in in vivo and SCNT blastocysts with/without Sc. IGF2 and H19 genes tended to be highly expressed in both SCNT blastocysts with (1.6-fold and 3.1-fold, respectively) and without (2.0-fold and 5.8-fold, respectively) Sc than that of the in vivo blastocysts. We found differences in imprinted gene expression patterns between in vivo and cloned blastocysts. Expression of H19 and IGF2 in SCNT blastocysts after Scriptaid treatment decreased towards the expression levels of in vivo blastocysts. These results indicated that Scriptaid treatment in SCNT embryos may also have beneficial effects on in vitro developmental competence as well as their gene expression pattern.

scholarly journals In vivo and in vitro maturation of rabbit oocytes differently affects the gene expression profile, mitochondrial distribution, apoptosis and early embryo development

2017 ◽  
Vol 29 (9) ◽  
pp. 1667 ◽  
Author(s):  
M. Arias-Álvarez ◽  
R. M. García-García ◽  
J. López-Tello ◽  
P. G. Rebollar ◽  
A. Gutiérrez-Adán ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rabbit Model ◽  
Developmental Competence ◽  
Gene Markers ◽  
Developmental Potential ◽  
Junction Protein ◽  
Mitochondrial Distribution ◽  
Potential Biomarkers

In vivo-matured cumulus–oocyte complexes are valuable models in which to assess potential biomarkers of rabbit oocyte quality that contribute to enhanced IVM systems. In the present study we compared some gene markers of oocytes and cumulus cells (CCs) from immature, in vivo-matured and IVM oocytes. Moreover, apoptosis in CCs, nuclear maturation, mitochondrial reallocation and the developmental potential of oocytes after IVF were assessed. In relation to cumulus expansion, gene expression of gap junction protein, alpha 1, 43 kDa (Gja1) and prostaglandin-endoperoxide synthase 2 (Ptgs2) was significantly lower in CCs after in vivo maturation than IVM. In addition, there were differences in gene expression after in vivo maturation versus IVM in both oocytes and CCs for genes related to cell cycle regulation and apoptosis (V-Akt murine thymoma viral oncogene homologue 1 (Akt1), tumour protein 53 (Tp53), caspase 3, apoptosis-related cysteine protease (Casp3)), oxidative response (superoxide dismutase 2, mitochondrial (Sod2)) and metabolism (glucose-6-phosphate dehydrogenase (G6pd), glyceraldehyde-3-phosphate dehydrogenase (Gapdh)). In vivo-matured CCs had a lower apoptosis rate than IVM and immature CCs. Meiotic progression, mitochondrial migration to the periphery and developmental competence were higher for in vivo-matured than IVM oocytes. In conclusion, differences in oocyte developmental capacity after IVM or in vivo maturation are accompanied by significant changes in transcript abundance in oocytes and their surrounding CCs, meiotic rate, mitochondrial distribution and apoptotic index. Some of the genes investigated, such as Gja1, could be potential biomarkers for oocyte developmental competence in the rabbit model, helping improve in vitro culture systems in these species.

scholarly journals Developmental and molecular correlates of bovine preimplantation embryos

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 895-904 ◽  
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.

scholarly journals Vitrification Effects on the Transcriptome of in vivo-Derived Porcine Morulae

2021 ◽  
Vol 8 ◽  
Author(s):  
Cristina Cuello ◽  
Cristina A. Martinez ◽  
Josep M. Cambra ◽  
Alejandro González-Plaza ◽  
Inmaculada Parrilla ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Pathways ◽  
Survival Rates ◽  
Gene Expression Pattern ◽  
Control Group ◽  
High Survival ◽  
Embryo Survival ◽  
Developmental Potential

Despite the reported promising farrowing rates after non-surgical and surgical transfers of vitrified porcine morulae and blastocysts produced in vivo (range: 70–75%), the pregnancy loss is 5–15 fold higher with vitrified than with fresh embryos. The present study aimed to investigate whether vitrification affects the transcriptome of porcine morulae, using microarrays and RT-qPCR validation. Morulae were obtained surgically from weaned sows (n = 13) on day 6 (day 0 = estrus onset). A total of 60 morulae were vitrified (treatment group). After 1 week of storage, the vitrified morulae were warmed. Vitrified-warmed and non-vitrified fresh morulae (control; n = 40) were cultured for 24 h to assess embryo survival by stereomicroscopy after. A total of 30 vitrified/warmed embryos that were deemed viable and 30 fresh control embryos (three pools of 10 for each experimental group) were selected for microarray analysis. Gene expression was assessed with a GeneChip® Porcine Genome Array (Affymetrix). An ANOVA analysis p-unadjusted &lt;0.05 and a fold change cut-off of ±1.5 were set to identify differentially expressed genes (DEGs). Data analysis and biological interpretation were performed using the Partek Genomic Suite 7.0 software. The survival rate of morulae after vitrification and warming (92.0 ± 8.3%) was similar to that of the control (100%). A total of 233 DEGs were identified in vitrified morulae (38 upregulated and 195 downregulated), compared to the control group. Nine pathways were significantly modified. Go-enrichment analysis revealed that DEGs were mainly related to the Biological Process functional group. Up-regulated DEGs were involved in glycosaminoglycan degradation, metabolic pathways and tryptophan metabolism KEGG pathways. The pathways related to the down-regulated DEGs were glycolysis/gluconeogenesis, protein export and fatty acid elongation. The disruption of metabolic pathways in morulae could be related to impaired embryo quality and developmental potential, despite the relatively high survival rates after warming observed in vitro. In conclusion, vitrification altered the gene expression pattern of porcine morulae produced in vivo, generating alterations in the transcriptome that may interfere with subsequent embryo development and pregnancy after embryo transfer.

Recruited vs. nonrecruited molecular signatures of brown, “brite,” and white adipose tissues

2012 ◽  
Vol 302 (1) ◽  
pp. E19-E31 ◽  
Author(s):  
Tomas B. Waldén ◽  
Ida R. Hansen ◽  
James A. Timmons ◽  
Barbara Cannon ◽  
Jan Nedergaard
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Expression Patterns ◽  
Gene Expression Pattern ◽  
Gene Expression Patterns ◽  
Culture Studies ◽  
Gene Markers ◽  
Different Types ◽  
Regulatory Functions

Mainly from cell culture studies, a series of genes that have been suggested to be characteristic of different types of adipocytes have been identified. Here we have examined gene expression patterns in nine defined adipose depots: interscapular BAT, cervical BAT, axillary BAT, mediastinic BAT, cardiac WAT, inguinal WAT, retroperitoneal WAT, mesenteric WAT, and epididymal WAT. We found that each depot displayed a distinct gene expression fingerprint but that three major types of depots were identifiable: the brown, the brite, and the white. Although differences in gene expression pattern were generally quantitative, some gene markers showed, even in vivo, remarkable depot specificities: Zic1 for the classical BAT depots, Hoxc9 for the brite depots, Hoxc8 for the brite and white in contrast to the brown, and Tcf21 for the white depots. The effect of physiologically induced recruitment of thermogenic function (cold acclimation) on the expression pattern of the genes was quantified; in general, the depot pattern dominated over the recruitment effects. The significance of the gene expression patterns for classifying the depots and for understanding the developmental background of the depots is discussed, as are the possible regulatory functions of the genes.

Splitting of IVP bovine blastocyst affects morphology and gene expression of resulting demi-embryos during in vitro culture and in vivo elongation

Zygote ◽  
2014 ◽  
Vol 24 (1) ◽  
pp. 18-30 ◽  
Author(s):  
Alejandra E. Velasquez ◽  
Fidel O. Castro ◽  
Daniel Veraguas ◽  
Jose F. Cox ◽  
Evelyn Lara ◽  
...  
Keyword(s):  
Gene Expression ◽  
Survival Rate ◽  
In Vitro Culture ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryo Morphology ◽  
Developmental Potential ◽  
Bovine Blastocyst

SummaryEmbryo splitting might be used to increase offspring yield and for molecular analysis of embryo competence. How splitting affects developmental potential of embryos is unknown. This research aimed to study the effect of bovine blastocyst splitting on morphological and gene expression homogeneity of demi-embryos and on embryo competence during elongation. Grade I bovine blastocyst produced in vitro were split into halves and distributed in nine groups (3 × 3 setting according to age and stage before splitting; age: days 7–9; stage: early, expanded and hatched blastocysts). Homogeneity and survival rate in vitro after splitting (12 h, days 10 and 13) and the effect of splitting on embryo development at elongation after embryo transfer (day 17) were assessed morphologically and by RT-qPCR. The genes analysed were OCT4, SOX2, NANOG, CDX2, TP1, TKDP1, EOMES, and BAX. Approximately 90% of split embryos had a well conserved defined inner cell mass (ICM), 70% of the halves had similar size with no differences in gene expression 12 h after splitting. Split embryos cultured further conserved normal and comparable morphology at day 10 of development; this situation changes at day 13 when embryo morphology and gene expression differed markedly among demi-embryos. Split and non-split blastocysts were transferred to recipient cows and were recovered at day 17. Fifty per cent of non-split embryos were larger than 100 mm (33% for split embryos). OCT4, SOX2, TP1 and EOMES levels were down-regulated in elongated embryos derived from split blastocysts. In conclusion, splitting day-8 blastocysts yields homogenous demi-embryos in terms of developmental capability and gene expression, but the initiation of the filamentous stage seems to be affected by the splitting.

Concurrence between the gene expression pattern of Actinobacillus actinomycetemcomitans in localized aggressive periodontitis and in human epithelial cells

2005 ◽  
Vol 54 (5) ◽  
pp. 497-504 ◽  
Author(s):  
Joseph Richardson ◽  
Justin Corey Craighead ◽  
Sam Linsen Cao ◽  
Martin Handfield
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Expression Pattern ◽  
Gene Expression Pattern ◽  
Aggressive Periodontitis ◽  
Actinobacillus Actinomycetemcomitans ◽  
Bacterial Gene ◽  
Bacterial Gene Expression

Actinobacillus actinomycetemcomitans is a facultatively intracellular pathogen and the aetiological agent of localized aggressive periodontitis. Screening of the genome of A. actinomycetemcomitans for in vivo-induced antigen determinants previously demonstrated that the proteome of this organism differs in laboratory culture compared with conditions found during active infection. The aim of the present study was to determine whether the bacterial gene expression pattern inferred with in vivo-induced antigen technology (IVIAT) in human infections was consistent with the gene expression pattern occurring upon epithelial cell association. To this end, a real-time PCR method was developed and used to quantify absolute and relative bacterial gene expression of A. actinomycetemcomitans grown extra- and intracellularly in two human epithelial cell lines (HeLa and IHGK). The amount of template used in the assay was normalized using the total count of viable bacteria (c.f.u.) as a reference point and performed in duplicate in at least two independent experiments. Controls for this experiment included 16S rRNA and gapdh. Transcription of all eight ORFs tested increased significantly (P < 0.05) in HeLa and IHGK cells compared with bacteria grown extracellularly. The concurrence of gene expression patterns found in the two models suggests that these epithelial cells are valid in vitro models of infection for the genes tested. IVIAT is an experimental platform that can be used as a validation tool to assess the reliability of animal and other models of infection and is applicable to most pathogens.

53 EFFECT OF ADDITION OF L-CARNITINE AND CONJUGATED LINOLEIC ACID DURING BOVINE EMBRYO CULTURE ON CRYOSURVIVAL, LIPID CONTENT, AND GENE EXPRESSION

2015 ◽  
Vol 27 (1) ◽  
pp. 119
Author(s):  
A. Ruiz ◽  
P. J. Hansen ◽  
J. Block
Keyword(s):  
Gene Expression ◽  
Lipid Metabolism ◽  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Lipid Content ◽  
Embryo Culture ◽  
Blastocyst Stage ◽  
Hatching Rate ◽  
Bovine Embryo

The objective was to determine the effects of addition of l-carnitine (LC) and trans-10, cis-12 conjugated linoleic acid (CLA) during bovine embryo culture on cryosurvival, lipid content, and gene expression. For all experiments, embryos were produced in vitro using abattoir-derived oocytes. Following insemination, presumptive zygotes were randomly assigned in a 2 × 2 factorial to be cultured in SOF-BE1 supplemented with or without 3.03 mM LC and 100 μM CLA until Day 7. For Exp. 1, blastocyst- and expanded-blastocyst-stage embryos (n = 777) were slow-frozen in 1.5 M ethylene glycol. Embryos were thawed and then cultured for 72 h. Re-expansion and hatching rates were recorded at 24, 48, and 72 h. There was no effect of LC on post-thaw re-expansion rates, but CLA reduced (P < 0.05) and tended (P < 0.08) to reduce re-expansion rate at 24 and 48 h, respectively (76.5 ± 2.5 v. 70.4 ± 2.5 and 79.5 ± 2.2 v. 76.0 ± 2.2, respectively). Whereas hatching rate at 72 h tended (P < 0.08) to be higher for embryos cultured with LC (67.8 ± 2.5 v. 74.4 ± 2.5), treatment with CLA reduced (P < 0.05) hatching rate at 48 h (62.3 ± 2.6 v. 54.9 ± 2.6). In Exp. 2, to determine lipid content, expanded blastocyst-stage embryos (n = 263) were harvested and stained using Nile Red. Embryos were examined for fluorescence using an epifluorescence microscope, and intensity of fluorescence per unit area was quantified using ImageJ software (NIH, Bethesda, MD, USA). There was a significant interaction (P < 0.01) between LC and CLA affecting embryo lipid content. Whereas addition of CLA during culture increased lipid, treatment with LC and the combination of LC and CLA reduced lipid (22.8 ± 1.1 v. 19.1 ± 1.1 v. 28.4 ± 1.1 v. 19.2 ± 1.2 for no additive, +LC, +CLA, and +LC and CLA, respectively). For Exp. 3, the effect of LC and CLA on the relative abundance of genes involved in lipid metabolism (ELOVL6, SCD1, SQLE, HMGCS1, CYP51A1, FDPS, FDFT1, LDLR, and SC4MOL) was determined. Pools of 5 expanded blastocyst-stage embryos from each treatment were collected across 5 replicates. The RNA was purified and synthesised into cDNA for RT-qPCR analysis. The SDHA, GAPDH, and YWAZ were used as housekeeping genes. Addition of LC during culture reduced (P < 0.05) the abundance of 4 of the 9 genes analysed (SQLE, HMGCS1, CYP51A1, and FDPS) and tended (P < 0.08) to reduce a fifth (FDFT1). In addition, there was a tendency (P < 0.08) for LC to increase the abundance of SCD1. Addition of CLA during culture had minimal effects on transcript abundance. In particular, CLA treatment reduced (P < 0.01) ELOVL6 and tended (P < 0.08) to increase SCD1. In contrast to previous studies, post-thaw cryosurvival was not significantly improved by treatment with LC or CLA. Results indicate that reduced embryo lipid content caused by LC treatment is due, in part, to an alteration in the abundance of genes involved in lipid metabolism. Further research is still necessary to determine whether in vivo survival following transfer of cryopreserved embryos can be enhanced by treatment with LC or CLA.Support was provided by USDA AFRI Grant 2010–85122–20623.

58 HISTONE DEACETYLASES INHIBITOR, SCRIPTAID, IS BENEFICIAL TO THE REPROGRAMMING OF SOMATIC NUCLEI FOLLOWING NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 129
Author(s):  
J. G. Zhao ◽  
J. W. Ross ◽  
Y. H. Hao ◽  
D. M. Wax ◽  
L. D. Spate ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylases ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Untreated Group ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology with potential applications in both agriculture and regenerative medicine. The reprogramming of differentiated somatic nuclei into totipotent embryonic state following NT is not efficient and the mechanism is currently unknown. However, accumulating evidence suggests that faulty epigenetic reprogramming is likely to be the major cause of low success rates observed in all mammals produced through SCNT. It has been demonstrated that increased histone acetylation in reconstructed embryos by applying histone deacetylases inhibitor (HDACi) such as trychostatin A (TSA) significantly enhanced the developmental competence in several species in vitro and in vivo. However TSA has been known to be teratogenic. Compared with TSA, Scriptaid is a low toxic but more efficient HDACi (Su GH et al. 2000 Cancer Res. 60, 3137–3142). The objectives of this study were: 1) to investigate and optimize the application Scriptaid to the NT using Landrace fetal fibroblast cells (FFCs) as donor; 2) investigate the effect of increased histone acetylation on the developmental competence of reconstructed embryos from NIH mini inbred FFCs in vitro and in vivo. The reconstructed embryos were treated with Scriptaid at different concentrations (0 nm, 250 nm, 500 nm and 1000 nm) after activation for 14 to 16 h. IVF embryos without treatment were produced as an additional control. Developmental rates to the 2-cell and blastocyst stage were determined. Developmental potential was determined by transferring Day 1 NT zygotes to the oviducts of surrogates on the day of, or one day after, the onset of estrus. Experiments were repeated at least 3 times and data were analyzed with chi-square tests using SAS 6.12 program (SAS institute, Inc., Cary, NC, USA). The percentage blastocyst of cloned embryos using Landrace FFCs as donors treated with 500 nm Scriptaid was the highest and was significantly higher than untreated group (25% v. 11%, P < 0.05). Percent cleaved was not different among four treatment groups. We used 500 nm Scriptaid for 14 to 16 h after activation for all subsequent experiments. Developmental rate to the blastocyst stage was significantly increased in cloned embryos derived from NIH mini inbred FFCs after treating with Scriptaid (21% v. 9%, P < 0.05), while the blastocyst rate in IVF group was 30%. Embryo transfer (ET) results showed that 5/6 (Transferred embryos No. were 190, 109, 154, 174, 152, and 190, respectively) surrogates (83%) became pregnant resulting in 2 healthy piglets from 2 litters (recipients received 190 and 154 embryos, respectively) in the Scriptaid treatment group, while no pregnancies were obtained in the untreated group from 5 ET (Embryos transferred No. are 140, 163, 161, 151 and 151, respectively). These results suggest that 500 nm Scriptaid treatment following activation increase both the in vitro and in vivo development of porcine SCNT embryos from NIH mini inbred FFCs and the hyperacetylation might actually improve reprogramming of the somatic nuclei after NT. Funding from the National Institutes of Health National Center for Research Resources RR018877.

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