bluuebunny/crossref_metadata_2025 huggingface dataset:
193 DIFFERENTIAL EFFECTS OF CULTURE ON APOPTOTIC GENE EXPRESSION IN THE PRE-IMPLANTATION CLONED EMBRYOS OF MINIATURE PIG
Authors: Park M. R. , Hwang I. S. , Moon H. J. , Shim J. H. , Kim D. H. , Yang B. C. , Cheong H. J. , Ko Y. G. , Yang B. S. , Im G. S.
Published: December 2006

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.
196 A NEW APPROACH TO PREDICT MOUSE EMBRYONIC DEVELOPMENTAL COMPETENCE USING A TIME-LAPSE SYSTEM AND THE 'SHORTEST HALF' analysis procedure
Authors: Yavin S. , Aroyo A. , Roth Z. , Arav A.
Published: December 2006

Embryonic development is a dynamic process in which embryo morphology may change immensely within several hours. Therefore, identifying and selecting embryos with the highest probability of developing and achieving a pregnancy is a major challenge. The timing of embryonic cleavage may serve as an additional indicator for the identification of quality embryos. The aim of this study was to characterize the cleavage timing of mouse embryos and to identify the stage that is most indicative of blastocyst formation. Mated mice (CB6F1) were sacrificed 20 h after hCG administration; putative zygotes were recovered and cultured (50 embryos in each 20-µL drop of M16) in a time-lapse system (EmbryoGuard; IMT, Ltd., Ness-Ziona, Israel) inside the incubator. The time-lapse system was programmed to take photos at half-hour intervals such that culture dishes were not removed from the incubator. The ‘shortest half’ statistical procedure of JMPIN (SAS Institute, Inc., Cary, NC, USA) was utilized to evaluate the period during which at least 50% of the embryonic population cleaves within the shortest time frame. Captured images made it possible to search along the time axis for the densest 50% of cleavage observations. Developing embryos were categorized into 3 groups according to the time of cleavage after hCG administration: before, during, and after the ‘shortest half’ for each developmental stage. Two hundred thirty putative zygotes cleaved and created 2-cell-stage embryos, of which 55 arrested at various stages and 175 progressed to the blastocyst stage. During embryonic development, cleavage timing appeared to become less uniform and the ‘shortest half’ became longer for each successive cell division: Whereas the shortest period in which 50% of the 2-cell-stage embryos cleaved was a 2-h interval, cleavage into the 4-cell, 8-cell, and blastocyst stages took 2.5, 3.5, and 5 h, respectively. The ‘short half’ for the first cleavage appears to be a predictive time frame for subsequent embryonic development, because cleavage was closely synchronized with 80% of the embryos developing to the blastocyst stage. Note that only a small number of embryos were actually cleaving early, while the ‘shortest half’ consisted of 50% of the embryonic population. Moreover, late-cleaving embryos in the 2-cell stage expressed inferior developmental potential relative to those that cleaved within the ‘shortest half’ (see Table 1). In summary, 2-cell-stage embryos that cleaved within the ‘shortest half’ seemed to be better synchronized and consequently more competent than the rest of the embryonic population. Embryonic cleavage timing using the ‘shortest half’ parameter can be considered a biological indicator of embryo potential. It may be useful as an additional tool for selecting embryos for transfer and cryopreservation.

Table 1.
Cleavage timing distribution into the 2-cell stage according to the shortest half
197 EFFECTS OF HYALURONAN ON OXYGEN CONSUMPTION AND ATP CONTENT IN PIG BLASTOCYSTS PRODUCED IN VITRO
Authors: Yoshioka K. , Yokoo M. , Ozawa T. , Suzuki C. , Abe H. , Rodríguez-Martínez H.
Published: December 2006

Hyaluronan (HA), a glycosaminoglycan present in follicular and oviductal fluids, has been related to sperm capacitation, fertilization, and embryo development. We have found that exogenous HA improves cell proliferation of porcine embryos cultured in a chemically defined medium (Yoshioka et al. 2004 Reprod. Fertil. Dev. 16, 264–265). Moreover, mitochondrial maturation was clearly more advanced in blastocysts cultured with HA compared to those cultured without HA, as seen by transmission electron microscopy. In the present study, the effects of HA on oxygen consumption and ATP content of blastocysts, produced in a defined system which reflects metabolic activity, were investigated. Porcine immature oocytes were matured for 44 h in porcine oocyte medium (POM) and subsequently fertilized with frozen–thawed ejaculated semen in porcine gamete medium supplemented with theophylline, adenosine, and cysteine (PGMtac4). Both POM and PGMtac4 were chemically defined media modified from porcine zygote medium (PZM)-5. After IVF, presumptive zygotes were cultured in PZM-5 containing HA (from the microorganism, Nacalai tesque, Kyoto, Japan) at concentrations of 0 [control], 10 [HA10], or 100 [HA100] �g mL-1 until 5 days after IVF. Blastocyst formation rate and total cell numbers/blastocyst at Day 5 were assessed. In addition, oxygen consumption and ATP content of single Day 5 blastocysts were measured. Blastocyst oxygen consumption was quantified using scanning electrochemical microscopy (HV-403; Research Institute for the Functional Peptides, Yamagata, Japan), and embryonic ATP content was determined using a commercial assay based on the luciferin-luciferase reaction (ATPlite; PerkinElmer, Groningen, The Netherlands). Data were statistically analyzed by ANOVA and Fisher's PLSD test. While the percentage of embryos that developed to the blastocyst stage [30.5% (63/206) to 31.7% (65/206)] did not differ among treatments, blastocyst cell number in the HA100 group [57.9 cells (n = 64)] was greater (P < 0.05) compared to those in the control [48.6 cells (n = 63)] or HA10 [50.0 cells (n = 65)] groups. Blastocyst oxygen consumption rate in the HA100 group [0.629 � 10-14 mol s-1 (n = 15)] was significantly higher than in the control [0.500 � 1-14 mol s-1 (n = 16)] or HA10 [0.464 � 10-14 mol s-1 (n = 14)] groups. ATP content/blastocyst did not differ among treatments [control: 0.645 pmol (n = 38), HA10: 0.727 pmol (n = 42), and HA100: 0.704 pmol (n = 43)]. It is concluded that HA affects the metabolic activity of pig blastocysts developed in a chemically defined medium, enhancing oxygen consumption and their total cell numbers, thus improving the quality of IVP blastocysts.
2 GLOBAL PEPTIDE SEQUENCING AND QUANTIFICATION OF PROTEINS IN PORCINE PARTHENOTES BY PROTEOMICS
Authors: Jang J. M. , Gupta M. K. , Jung J. W. , Uhm S. J. , Kim K. P. , Lee H. T.
Published: December 2006

Mass spectrometry (MS) is a powerful emerging tool in proteomics for global identification and quantification of proteins and their differential analysis. The aim of this study was to utilize MS for global peptide sequencing and the relative quantification of peptide sequences in porcine embryos produced by parthenogenesis. Oocytes (n = 6000) recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro for 42–44 h and parthenogenetically activated by a single electric pulse of 1.36 kV cm-1. After 24 h of in vitro culture in NCSU23 medium supplemented with 0.4% BSA, presumptive zygotes were sampled in Laemmli buffer and stored in liquid nitrogen until analysis. SDS-PAGE was used as the first step to separate intact proteins into 16 fractions. Each fraction was then in-gel tryptic-digested and analyzed by nanoLC-nano-ESI-MS/MS to sequence the peptide components in each fraction by LTQ ion-trap mass spectrometer (Finnigan, Fresno, CA, USA) after peptide purification by C18 ZipTips (Millipore, Billerica, MA, USA). Synthetic peptides from Myoglobin (LFTGHPETL*EK) and MAP3 Kinase (IPTGTV*HNQAK) protein having an isotope-labeled carbon (*) were used as internal standards for relative quantification. In total, 54 060 peptides were sequenced. Of these, 259 proteins were identified in the NCBI protein database for Sus scrofa using Spectrum Mill software (Agilent Technologies, Palo Alto, CA, USA). Of these, 199 proteins were identified with high confidence having at least 2 peptide sequences matching each protein. These included heat shock protein families, ribosomal proteins, cytoskeletal proteins, mitochondrial enzymes, disulfide isomerase proteins, glutathione S-transferase, DNA methyltransferase, STAT1, Ras-related protein Rab 1A, voltage-dependent ion channel proteins, zinc finger proteins and elongation factors, etc., which are involved in several important metabolic and cell signaling pathways. Several proteins including DNA methyltransferase 1, peroxidoxin 2, ubiquitin carboxyl-terminal hydrolase L1, serpin, heat shock proteins, cytoskeletal proteins, mitochondrial enzymes, elongation factor 1, bone morphogenetic protein, glucose-related proteins, etc., were among the highly abundant proteins. Our results thus showed that proteomic strategy can be successfully applied to analyze porcine embryo proteome. To our knowledge, this is the first time that several proteins have been sequenced and quantified in porcine parthenotes by proteomic means. In the long run, this study may help the understanding of mechanisms underlying embryonic development and identification of biomarkers of embryo quality.
This work was supported by grants from the Research Project on the Production of Bio-organs (No. 200503030202), Ministry of Agriculture and Forestry, and Korea Health 21 R&D Project (No. A060769), Ministry of Health and Welfare, Republic of Korea.
200 EFFECT OF OOCYTE SIZE AND MICROMANIPULATION TECHNIQUES (ICSI AND EMBRYO BIOPSY) ON GOAT EMBRYO QUALITY
Authors: Jimenez-Macedo A. R. , Anguita B. , Izquierdo D. , Romaguera R. , Morato R. , Mogas T. , Paramio M. T.
Published: December 2006

The aim of this study was to evaluate embryo quality of prepubertal goat oocytes after micromanipulation techniques: ICSI and embryo biopsy. Prepubertal goat oocytes were recovered from a slaughterhouse and matured in IVM medium (TCM-199 with serum, hormones, and cystamine) for 27 h. Spermatozoa were selected by swim-up and capacitated with heparin (10 µg mL−1) plus ionomycin (200 nM) for 15 min. A total of 930 IVM oocytes were divided into 4 groups depending on size (Group I: 110–125 µm, and Group II: >125 µm) and fertilization procedure: ICSI or IVF. Zygotes were cultured in TALP fertilization medium. At 24 h post-insemination (hpi), zygotes were cultured in SOF. At 48 hpi, oocyte cleavage was recorded. Four days after insemination, a sample of 8-cell embryos was biopsied by removal of one blastomere. After biopsy, embryos were cultured in SOF for a further 4 days. Blastocysts were stained by TUNEL to assess apoptotic cell percentage. Results are shown in Table 1. Biopsied and non-biopsied embryos from Group II had higher blastocyst rates than those in Group I both in ICSI and IVF (P ≤ 0.02). Group II had also larger blastocyst cell numbers than Group I both in ICSI and in IVF (P ≤ 0.001). Moreover, blastocysts from Group II non-biopsied embryos had statistically larger cell numbers than those from the biopsied ones (P ≤ 0.05). Blastocysts from Group II had a lower apoptotic rate than those in Group I both in ICSI and IVF (P ≤ 0.05). In conclusion, oocytes larger than 125 µm were more competent to achieve embryo development than smaller oocytes. ICSI did not reduce embryo quality, either in blastocyst cell number or apoptotic index, when compared with IVF. Furthermore, embryo biopsy did not reduce blastocyst rates or apoptotic index. However, embryo biopsy reduced blastocyst cell number in Group II-derived blastocysts.

Table 1.Effect of oocyte size and micromanipulation techniques on goat embryo quality
221 EPIGENETIC DYNAMICS OF PLURIPOTENCY GENES IN THE CONTEXT OF DIFFERENTIATION AND NUCLEAR REPROGRAMMING DETERMINED BY Q2ChIP, A QUICK AND QUANTITATIVE CHROMATIN IMMUNOPRECIPITATION TECHNIQUE APPLICABLE TO SMALL CELL SAMPLES
Authors: Dahl J. A. , Taranger C. K. , Collas P.
Published: December 2006

Interactions between proteins and DNA are essential for cellular functions such as genomic stability, DNA replication and repair, chromosome segregation, transcription, and epigenetic silencing of gene expression. Chromatin immunoprecipitation (ChIP) is a key technique for mapping histone modifications and transcription factor binding on DNA and thereby unraveling the role of epigenetics in the regulation of gene expression. Current ChIP protocols require extensive sample handling and large numbers of cells (5-10 million). primarily owing to ample loss of material during the procedure. We altered critical steps of conventional ChIP to develop a quick and quantitative (Q2) ChIP assay suitable for cell numbers 100- to 1000-fold lower than those required for conventional ChIP. Key modifications of the ChIP procedure include (i) formaldehyde DNA–protein cross-linking in suspended cells, (ii) cross-linking in the presence of 20 mM sodium butyrate to enhance specificity of precipitation of acetylated histones, (iii) transfer of washed precipitated immune complexes to a clean tube ('tube shift') to increase ChIP specificity by virtually eliminating nonspecifically bound chromatin, and (iv) combination of cross-link reversal, protein digestion, and DNA elution into a single 2-h step. We used Q2ChIP to monitor changes in 6 histone H3 modifications on the human developmentally regulated genes OCT4 (POU5F1), NANOG, and LMNA (lamin A) in the context of retinoic acid (RA)-mediated differentiation of embryonal carcinoma cells and upon reprogramming of kidney epithelial 293T cells to pluripotency in carcinoma cell extract (Taranger et al. 2005 Mol. Biol. Cell 16, 5719–5735). Real-time PCR analysis of precipitated DNA unravels an unexpected two-step heterochromatin assembly elicited by RA on the OCT4 proximal promoter, proximal enhancer, and distal enhancer, and on the NANOG promoter, whereby methylation of H3K9 and H3K27 is followed by H3K9 deacetylation. H3K4 di- and trimethylation remain relatively unaffected by RA treatment. In contrast, reprogramming of 293T cells in carcinoma extract promotes assembly of histone marks characteristic of transcriptional induction of OCT4 and NANOG, such as acetylation and demethylation of H3K9. The results argue toward ordered chromatin repackaging at developmentally regulated promoters upon differentiation or, conversely, nuclear reprogramming to pluripotency.
228 ESTABLISHMENT OF EMBRYONIC STEM-LIKE CELL LINES IN RABBITS
Authors: Ou Y.-W. , Lee K.-H. , Chen L.-R. , Tang P.-C. , Guu H.-F. , Chen M.-J. , Ju J.-C.
Published: December 2006

Embryonic stem (ES) cells are pluripotent cells from the inner cell mass (ICM) of the blastocyst. They are capable of differentiating to various cell types, such as neural cells, cardiocytes, hepatic cells, and germ cells. The aim of this study was to establish rabbit ES cell lines as an animal model for human diseases. Blastocysts were collected from New Zealand White rabbits during Days 4 to 5 after breeding. After removal of the mucin coat and the zona pellucida by pronase, the embryos were directly cultured in ES cell medium on mitomycin C-treated mouse embryonic fibroblast (MEF) or STO feeder layers. In Experiment 1, the efficiencies of 2 different feeder layers, MEF and STO, in generating rabbit ES cell lines were compared. Six blastocysts were used for each STO and MEF feeder group. The primary ICM colonies were formed in 67% (4/6) of the cultures on the STO and 83% (5/6) on the MEF. Sixty percent of those primary colonies (3/5) were successfully grown into ES-like cell lines in the MEF feeder group. However, no cell lines were established on the STO feeder. In Experiment 2, whole blastocysts or ICMs isolated by immunosurgery were cultured to establish ES cell lines. A total of 21 blastocysts were recovered from 2 does. Eighteen whole blastocysts and 3 isolated ICMs were cultured on the MEF feeders. Twelve (67%) of the cultured whole blastocysts formed primary ICM colonies, of which 5 (42%) of the cultures continuously propagated and formed ES-like cell lines. In the immunosurgical group, 2 of the 3 isolated ICMs formed primary colonies but only 1 ES-like cell line was established. A total of 9 ES-like cell lines maintained morphological undifferentiation after 14 passages and expressed alkaline phosphatase activity. Seven of the 9 ES-like cells expressed Oct-4 and the stage-specific embryonic antigen-4 (SSEA-4) as detected by immunocytochemical staining. Two cell lines were further induced to differentiate into embryoid bodies in suspension culture. Another 3 cell lines were injected into SCID mice and one of them formed a teratoma. The competence of generating chimeric rabbits and the teratogenicity of the established ES-like cell lines are under evaluation. In conclusion, rabbit ES-like cells were efficiently generated and whole-blastocyst culturing on the MEF feeder appeared to be a preferred method for the isolation and maintenance of rabbit ES-like cell lines.
23 HYPOMETHYLATION OF DNA IN NUCLEAR TRANSFER EMBRYOS FROM PORCINE EMBRYONIC GERM CELLS
Authors: Ahn K. S. , Heo S. Y. , Won J. Y. , Shim H.
Published: December 2006

Epigenetic modification including genome-wide DNA demethylation is essential for normal embryonic development. Insufficient demethylation of the somatic cell genome may cause various anomalies and prenatal loss in the development of nuclear transfer embryos. Species-specific differences in the epigenetic status of cloned donor genomes have been reported. A level of DNA methylation in porcine somatic cell nuclear transfer (SCNT) embryos was similar to that of normally fertilized embryos, but hypermethylation of DNA in bovine SCNT embryos was commonly observed (Kang et al. 2001 J. Biol. Chem. 276, 39 980-39 984). Even in the same species, the source of the nuclear donor often affects later development of nuclear transfer embryos. In this study, appropriateness of porcine embryonic germ (EG) cells as karyoplasts for nuclear transfer with respect to epigenetic modification was investigated. These cells follow the methylation status of the primordial germ cells from which they originated, so they may contain a less methylated genome than somatic cells. The rates of blastocyst development were similar among embryos from EG cell nuclear transfer (EGCNT), SCNT, and intracytoplasmic sperm injection (ICSI) (16/62, 25.8% vs. 56/274, 20.4% vs. 16/74, 21.6%, respectively). Genomic DNA samples from EG cells (n = 3), fetal fibroblasts (n = 4), and blastocysts from EGCNT (n = 8), SCNT (n = 14), and ICSI (n = 6) were isolated and treated with sodium bisulfite. The satellite region (GenBank Z75640) that involves 9 selected CpG sites was amplified by PCR, and the rates of DNA methylation in each site were measured by pyrosequencing technique (Biotage AB, Uppsala, Sweden). The average methylation degrees of CpG sites in EG cells, fetal fibroblasts, and blastocysts from EGCNT, SCNT, and ICSI were 17.9, 37.7, 4.1, 9.8, and 8.9%, respectively. The genome of porcine EG cells was less methylated than that of somatic cells (P < 0.05), and DNA demethylation occurred in embryos from both EGCNT (P < 0.05) and SCNT (P < 0.01). However, the degree of DNA methylation in EGCNT embryos was approximately one-half that of SCNT (P < 0.01) and ICSI (P < 0.05) embryos; in SCNT and ICSI embryos, the genome was demethylated to the same degree. The present study demonstrated that porcine EG cell nuclear transfer results in hypomethylation of DNA in cloned embryos, yet leading to normal pre-implantation development. However, it would be interesting to further investigate whether such modification affects long-term survival of cloned embryos.
230 GENERATION OF PUTATIVE RABBIT EMBRYONIC STEM CELLS
Authors: Wang S. , Tang X. , Niu Y. , Chen H. , Li T. , Tan T. , Ji W.
Published: December 2006

The rabbit, as a laboratory animal model, has several advantages in the study of human physiological disorders. In this study, stable putative pluripotent rabbit embryonic stem cells (rESCs) were derived from in vivo-fertilized and in vitro-cultured blastocysts. The rabbit ICMs were obtained by 0.05% trypsin–0.008% EDTA treatment and mechanical separation; the ES-like cell colonies seen several days later. ICM-derived outgrowths which were treated with 5 mg/mL-1 dispase, followed by 0.05% trypsin–0.008% EDTA, were mechanically disaggregated into small clumps and reseeded on MEFs. The putative ES cell lines maintained expression of pluripotent cells markers and normal XY karyotype for long periods of culture (>1 month). The putative rESCs expressed alkaline phosphatase, transcription factor Oct-4, stage-specific embryonic antigens (SSEA-1, SSEA-3, and SSEA-4), and tumor-related antigens (TRA-1-60 and TRA-1-81). The morphological characteristics of the putative ESCs are closer to those of human ESCs; their high speed of proliferation, however, is closer to that of mouse ESCs. Putative rabbit ESCs were induced to differentiate into many cell types including trophoblast cells, similar to primate ESCs, in vitro, and formed teratomas with derivatives of the 3 major germ layers in vivo when injected into SCID mice. Using RT-PCR measurement, but with some differences in ligands and inhibitors, and comparing with human and mouse ESCs, the putative rabbit ESCs expressed similar genes related to pluripotency (Oct-4, Nanog, SOX2, and UTF-1) and similar genes of FGF, WNT, and TGF signaling pathways related to the proliferation and self-renewal. Our further research work showed that TGF beta and FGF pathways cooperate to maintain pluripotency of rabbit ESCs similar to those of human ES cells.
239 EMBRYOTOXICITY ASSAY FOR ANTIVIRAL COMPOUND BPIP (5-[(4-BROMOPHENYL)METHYL]-2-PHENYL-5H-IMIDAZO[4,5-c]PYRIDINE) IN BOVINE IN VITRO-PRODUCED EMBRYOS
Authors: Mestach J. , Paeshuyse J. , Neyts J. , Nauwynck H. J. , Maes D. , Van Soom A.
Published: December 2006

Bovine viral diarrhea virus (BVDV) causes serious economic losses in the cattle industry. Evidence exists that only zona pellucida (ZP)-free bovine embryos are susceptible to BVDV infection (Vanroose et al. 1998 Biol. Reprod. 58, 857–866); however, BVDV can adhere to and therefore ‘infect’ both in vivo-(Waldrop et al. 2004 Theriogenology 62, 387–397) and in vitro-produced ZP intact embryos (Stringfellow et al. 2000 Theriogenology 53, 827–839). To eliminate these sanitary risks, pre-treatment of embryos with antiviral compounds may be a promising approach (Givens et al. 2006 Theriogenology 65, 344–355). BPIP (5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine) has been reported to display antiviral activity against BVDV, with a 50% effective inhibition of BVDV-induced cytopathic effect formation at a concentration of 0.04 µM (Paeshuyse et al. 2006 J. Virol. 80, 149–160). However, since the short- and long-term effects of BPIP have not been described, the aim of the current study was to assess whether addition of BPIP for 2 days at a concentration of 5 µM is toxic for ZP-free cattle embryos. Oocytes were aspirated from 3–6-mm follicles of cattle ovaries, matured for 24 h, and subsequently co-incubated with 1 × 106 sperm cells mL−1 in IVF-TALP with 20 µg/mL−1 heparin for 24 h at 39°C and 5% CO2 in air. After fertilization, presumptive zygotes were put in groups of 25 into 50-µL droplets of SOF under oil in 5% CO2, 5% O2, and 90% N2 for 6 days. Afterwards, morulae and blastocysts were collected, rendered ZP-free by means of pronase treatment, and divided into 4 groups: (i) ZP-free control group, (ii) ZP-free control group treated with a volume of DMSO equal to condition (iv), (iii) ZP-free group treated with 5 µM BPIP in DMSO, and (iv) ZP-free group treated with 10 µM BPIP in DMSO. Because BPIP is a fat-soluble molecule, embryos were cultured in 0.5 mL SOF without oil for 2 days. At Day 8, all embryos were fixed, TUNEL-stained, and analyzed for total cell number and percentage of apoptotic cells. Three independent replicates were performed. Results are shown in Table 1 and were analyzed by means of ANOVA. Only group iv showed a significant decrease in total cell number, indicating that at 10 µM BPIP may negatively influence embryo development. At both 5 and 10 µM, BPIP treatment resulted in an increase in percentage of apoptotic cells compared to the control group. However, a similar increase was observed using DMSO alone (group ii), indicating that the apoptotic effect may be due solely to the DMSO. In conclusion, BPIP does not appear to cause embryo toxicity at 5 µM, but an alternative, less toxic, dissolving agent may be considered.

Table 1.Embryotoxicity assay of BPIP
24 EFFECT OF TREATMENT WITH TRICHOSTATIN A ON IN VITRO DEVELOPMENT OF BOVINE NUCLEAR-TRANSFERRED EMBRYOS
Authors: Akagi S. , Fukunari K. , Matsukawa K. , Watanabe S. , Takahashi S.
Published: December 2006

It has been reported that 5 or 50 nM trichostatin A (TSA) treatment after somatic cell nuclear transfer (NT) improves the success rate of mouse cloning (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189). In this study, we examined the effect of TSA treatment on the in vitro development of bovine NT embryos. As donor cells for NT, bovine fibroblast cells of passages 3 to 5 were used following culture in serum-starved medium for 5 to 7 days. Oocytes were enucleated after in vitro maturation in TCM-199 supplemented with 10% fetal bovine serum. Enucleated MII oocytes were fused with fibroblast cells by a DC pulse of 25 V/150 µm for 10 µs in Zimmerman mammalian cell fusion medium. Fused oocytes were activated by 10 µM calcium ionophore for 5 min, followed by incubation with 2.5 µg mL−1 cytochalasin D, 10 µg mL−1 cycloheximide, and 5 or 50 nM TSA for 1 h, and then cycloheximide and 5 or 50 nM TSA for 4 h. After chemical activation, NT embryos were cultured in IVD-101 (Research Institute of Functional Peptide Co., Ltd., Yamagata, Japan) with 5 or 50 nM TSA for 10 h and subsequently cultured in IVD-101 without TSA. Control NT embryos were cultured in the same medium without TSA after fusion. After in vitro culture for 8 days, blastocyst formation and cell numbers of blastocysts were examined. The fusion rate of enucleated oocytes with fibroblast cells was 81% (199/247). In vitro development of NT embryos is summarized in Table 1. There were no differences in the cleavage rate and development rate to the blastocyst stage of NT embryos among control, and 5 and 50 nM TSA treatments. The cell number of 50 nM TSA-treated NT embryos at the blastocyst stage was higher than that of control NT embryos without TSA treatment. In conclusion, 50 nM TSA treatment for 15 h after activation did not affect the in vitro developmental competence, but increased total cell number in bovine NT embryos. These results suggest that TSA treatment may improve the quality of blastocysts in bovine NT.

Table 1.
Effects of TSA treatment on in vitro development of NT embryos derived from fibroblast cells
242 EFFECT OF SPERMATOZOA HEAD MORPHOMETRIC DIMENSIONS ON FREEZABILITY IN BROWN BEAR (URSUS ARCTOS)
Authors: Anel L. , Garcia-Macias V. , Martinez-Pastor F. , Alvarez M. , Borragan S. , Bernardo J. , Alves S. , Chamorro C. , Paz P.
Published: December 2006

Having recently observed that survival of red deer spermatozoa after cryopreservation seemed to reflect the size of sperm heads, we hypothesized that cryoresistance of brown bear spermatozoa might also be dependent on head size, since in a preliminary study we had also observed significant differences in sperm head sizes among male brown bears (median values for area 22.2 �m2, perimeter 18.2, length 6.1 and width 4.4 �m). In the present report, we analyzed the post-thaw survival of spermatozoa of 6 brown bears that were assigned to 2 groups (3 bears/group) based on sperm size: Group A with large-size sperm heads; Group B with small-size heads. Ejaculates were obtained by electroejaculation of adult brown bears (semi-free ranging in Cabarceno Park, Cantabria, Spain) under general anesthesia (7 mg kg-1 tiletamine + zolazepan and 2 mg kg-1 ketamine). Semen was diluted (Tes-Tris-fructose, 8% glycerol, 20% egg yolk, EDTA, and Equex paste), loaded in 0.25-mL straws, and frozen in a biofreezer at 20�C min-1 to -100�C. After storage in liquid nitrogen, samples were thawed in water at 65�C for 6 s and survival was measured. Sperm motility (TM: total, and PM: progressive; %) was assessed microscopically, and sperm viability, acrosome integrity (PI/PNA-FITC), and mitochondrial status (JC-1) were assayed for fresh and thawed sperm by flow cytometry. Recovery rates (RR: thawed/fresh � 100) were calculated for all parameters. For measurement of head size, fresh sperm samples were fixed in glutaraldehyde and slides were air-dried for 2 h. The samples were then stained with Diff-Quik� staining at 37�C. The area (Ar), perimeter (P), length (L), and width (W) of the heads of >100 spermatozoa per slide were measured (Sperm Class Analyzer�; Microptic S.L., Barcelona, Spain). Data were analyzed with the SAS ver8 system, and the Wilcoxon test was applied. The respective morphometric dimensions of the 2 groups were practically identical (Ar = 22; P = 18; L = 6; W = 4). The post-thaw recovery rates of spermatozoa from Group A were: TM: 60.1 � 29.3; PM: 54.8 � 36.0; viability: 99.4 � 8.0; acrosomes: 96.2 � 3.1; mitochondria: 70.9 � 15.5. The recovery rates for Group B were: TM: 78.7 � 13.8; PM: 69.0 � 18.8; viability: 93.8 � 5.2; acrosomes: 98.2 � 9.8; mitochondria: 72.5 � 22.5. Because of the high variability of recovery rates between males within each group, there were no statistical differences between the 2 groups. The absence of differences can be explained by the small number of males examined and the high variability between them. More studies are necessary to determine whether large sperm cells of brown bears are more susceptible to damage during cryopreservation.
This work was supported in part by CANTUR S.A. and CICYT (CGL 2004-0278/BOS).
245 USE OF A TRIPLE STAIN (SYBR-14/PI/MC540) FOR VIABILITY AND CAPACITATION ASSESSMENT IN THAWED SEMEN FROM BROWN BEAR (URSUS ARCTOS)
Authors: Garcia-Macias V. , Martinez-Pastor F. , Alvarez M. , Paz P. , Borragan S. , Anel E. , Mata M. , Nicolas M. , Anel L.
Published: December 2006

Application of new sperm assessment techniques would improve our capability to determine the effects of cryopreservation on sperm function. This becomes relevant when germplasm banks are established for endangered species, as in the case of the brown bear in Spain. Different triple stain techniques have been used in conjunction with flow cytometry to assess various sperm attributes including viability and acrosome status. However, fluorochromes with similar emission spectra may interfere with data resolution, making acquisition and interpretation of data difficult. The double stain combination of SYBR-14 and PI (propidium iodide; max λ 617) has been widely used to differentiate live from dead spermatozoa (spz), and, more recently, merocyanine 540 (MC; max λ 555) has been used to detect a sperm membrane lipid disorder associated with sperm capacitation. In the present study, we analyzed the suitability of combining SYBR-14/PI with MC for simultaneous determination of the viability and capacitation status of frozen–thawed spermatozoa of brown bears (n = 10; semi-free ranging; Cabarceno Park, Cantabria, Spain) obtained by electroejaculation under general anesthesia (7 mg kg-1 tiletamine + zolazepan and 2 mg kg-1 ketamine). Semen was diluted (Tes-Tris-fructose, 8% glycerol, 20% egg yolk, EDTA, and Equex paste), loaded in 0.25-mL straws, and frozen in a biofreezer at 20�C min-1 to -100�C. After storage in liquid nitrogen, samples were thawed at 65�C for 6 s, divided into 2 aliquots (1–2 million spz mL-1), extended with 300 �L PBS, and stained with SYBR14 (1.2 �L) and PI (3 �L; LIVE/DEAD� Sperm Viability Kit; Molecular Probes, Inc., Eugene, OR, USA). To evaluate the possible interaction of MC on sperm viability, half of the aliquots were counterstained with 1.5 �L of MC (diluted with 2.7 �M of DMSO); the other half were not counterstained (control). All tubes were incubated at 37�C for 30 min, and assessed by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA). Data were analyzed with Bland-Altman. Results indicated that MC staining was mainly confined to dead spermatozoa (22.2 � 7.7%), whereas a lower percentage of live spermatozoa (5.7 � 1.6%; P < 0.05) were also stained with MC. Possibly, the staining of dead spermatozoa with MC was due to capacitation changes induced by cryopreservation. The percentage of live spermatozoa was not different between samples counterstained with MC (68.9 � 9.2) and non-MC-stained control samples (68.6 � 8.8). Thus, we consider that MC does not influence SYBR14/PI discrimination of viable spermatozoa, and that the 3 stains can be used simultaneously. However, more studies are necessary to determine whether MC can be used to distinguish the capacitation status of brown bear thawed spermatozoa.
This work was supported by CANTUR S.A. and CICYT (CGL 2004-0278/BOS).
246 IN VITRO PRODUCTION OF SPRINGBOK (ANTIDORCAS MARSUPIALIS) EMBRYOS
Authors: Krisher R. , Auer A. , Clark K. , Emsweller K. , Rogers S. , Thomas K. , Chatiza F. , Bartels P.
Published: December 2006

The objective of this experiment was to develop in vitro embryo production (IVP) technologies in springbok (Antidorcas marsupialis), a southern African antelope. Springbok, a fairly common species on game farms in parts of South Africa, may be used as a model species for gamete rescue and IVP techniques to be applied to the conservation of other threatened antelope species. Springbok belong to the family bovidae, subfamily antilopinae, tribe antilopini, which comprises about twenty species in genera Gazella, Antilope, Procapra, Antidorcas, Litocranius, and Ammodorcas. In this tribe alone, there are 4 species or subspecies that are critically endangered, 3 that are endangered, and 10 that are considered vulnerable, demonstrating the need for antelope conservation efforts. In addition, our studies contributed to the South African biological resource bank, so that banked springbok semen and embryos might be used in the future for managed genetic contribution to isolated captive or wild populations via assisted reproductive technologies. Oocytes were recovered (3 replicates) from ovaries obtained at supervised culls for management purposes in South Africa, and cultured in defined Gmat or undefined TCM-199 with FCS maturation medium for 28-30 h (Brad et al. 2004 Reprod. Fertil. Dev. 16, 223). Oocytes were fertilized with frozen-thawed springbok epididymal spermatozoa in modified SOF fertilization medium with caffeine (Herrick et al. 2004 Biol. Reprod. 71, 948–958). Eighteen hours after insemination, a randomly selected subset of the zygotes were fixed to determine fertilization success. The remaining zygotes were cultured in G1/G2 media. On Day 7 of culture, embryos were analyzed for development to the morula or blastocyst stage. A total of 259 selected oocytes were collected from 50 females (5.2 selected oocytes/female on average). There was no difference in the percentage of oocytes normally fertilized (2 pronuclei, PN) between oocytes matured in Gmat (n= 43; 12%) and those matured in TCM-199 (n= 42; 10%). There were significantly (P < 0.05) more oocytes penetrated (e2 PN) when matured in TCM (50%) compared to Gmat (23%). There were no differences in embryonic cleavage or morula/blastocyst development (of total oocytes inseminated) between treatments (Gmat,n= 89, 54%, 9.0%; TCM-199, n= 85, 68%, 9.4%, respectively). In both treatments, the average blastocyst grade was 2.125 using the standard bovine grading system (Curtis, Cattle Embryo Transfer Procedure, 1991). In conclusion, in vitro oocyte maturation, fertilization, and embryo culture to the blastocyst stage is possible in springbok. Importantly, blastocysts can be produced in vitro under semi-defined conditions, demonstrating that oocyte maturation without serum does support developmental competence. This is important for the potential international movement of IVP embryos to be used for genetic management in the conservation of antelope species.
259 DNA HYPOMETHYLATION OF FETAL FIBROBLASTS IMPROVES DEVELOPMENTAL COMPETENCY AND GENE EXPRESSION PATTERN IN PORCINE EMBRYOS FOLLOWING NUCLEAR TRANSFER
Authors: Mohana Kumar B. , Jin H. F. , Kim J. G. , Ock S. A. , Song H. J. , Jeong Y. J. , Balasubramanian S. , Rho G. J.
Published: December 2006

The inhibition of methyl groups in the DNA of donor cells has been hypothesized to improve the potential reprogramming by the enucleated ooplasm after nuclear transfer (NT). Previously, we reported that treatment of porcine fetal fibroblasts (PFF) with an inhibitor of methylation, 5-azacytidine (5-azaC) at 0.5 �m, results in the retention of desirable characteristics with a relative reduction in methylation, making cells more conducive for reprogramming (Mohana Kumar et al. 2006 Cell Tissue Res. 325, 445-454). To understand these observations further, the present study investigated the developmental competence and expression pattern of gene transcripts in porcine NT embryos from PFF (control) and 0.5 �m 5-azaC-treated PFF (PFF + 5-azaC) at 4-cell, 8-cell, morula, and blastocyst stages, and compared these with those of IVF and in vivo embryos. Cleavage rate was significantly (P < 0.05) higher in IVF than in NT embryos from PFF and PFF + 5-azaC (86.7 � 5.2% vs. 65.8 � 5.3% and 69.3 � 4.4%, respectively). Similarly, significantly (P < 0.05) higher blastocyst rates were observed in IVF embryos (27.2 � 2.1%). However, NT embryos from PFF + 5-azaC showed enhanced developmental potential with significantly (P < 0.05) higher rates of blastocysts (21.3 � 2.2%) than NT embryos from PFF (14.8 � 1.9%). NT embryos from PFF + 5-azaC (33.8 � 4.1) had significantly (P < 0.05) higher total cell numbers than from PFF (24.6 � 3.5), but did not differ in the proportion of apoptotic cells (6.9 � 1.8% and 7.2 � 2.1%, respectively). However, the high total cell number and lower incidence of apoptosis were observed in IVF and in vivo embryos (45.3 � 3.8, 2.7 � 0.8%, and 53.9 � 3.5, 1.2 � 0.9%, respectively). Alterations in the expression pattern of genes implicated in transcription and pluripotency (Oct4 and Stat3), DNA methylation (DNA methyltransferases: Dnmt1, Dnmt2, Dnmt3a, and Dnmt3b), histone acetylation (histone acetyltransferase 1-HAT1), and histone deacetylation (histone deacetylases-Hdac1, Hdac2, and Hdac3) were observed in NT embryos from PFF and PFF + 5-azaC compared with that in IVF and in vivo counterparts. However, the expression of genes in PFF + 5-azaC-NT embryos closely followed those of in vivo-derived embryos compared with PFF-NT embryos, and, interestingly, there was lower variability in the expression of genes related to DNA methylation. Our findings demonstrate that remodeling of the epigenetic status by partial reduction of somatic DNA methylation from donor cells is beneficial in improving the developmental competency of porcine NT embryos. Further, hypomethylated donors may be more efficiently reprogrammed to re-activate the expression of early embryonic genes.
This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.
261 PHENOTYPIC CHARACTERISTICS AND TISSUE-SPECIFIC IGF2R/IGF2 EXPRESSION PARTITION BOVINE FETAL OVERGROWTH ASSOCIATED WITH IN VITRO FERTILIZATION AND SOMATIC CELL NUCLEAR TRANSFER CLONING
Authors: Bebbere D. , Ulbrich S. E. , Zakhartchenko V. , Weppert M. , Reichenbach H.-D. , Meyer H. H. D. , Ledda S. , Wolf E. , Hiendleder S.
Published: December 2006

Large offspring syndrome (LOS) in ruminants refers to various poorly defined organ pathologies that are associated with fetal overgrowth and are encountered after a range of embryo manipulations (Rhind et al. 2003 Nat. Rev. Genet. 4, 855–864). We have explored the effects of somatic cell nuclear transfer (NT) and in vitro fertilization (IVF) on phenotype and relative expression levels of 2 imprinted genes important for fetal growth, insulin-like growth factor 2 (IGF2) and its receptor (IGF2R). Viable bovine fetuses were recovered near the end of the first trimester of pregnancy, and skeletal muscle, liver, and lung were sampled for real-time RT-PCR analyses. We compared NT-fetuses (n = 23), IVF-fetuses (n = 24), and fetuses generated by artificial insemination (controls, n = 24) in order to separate abnormalities specific to cloning from effects of in vitro gamete and embryo manipulation. Nuclear transfer and IVF-fetuses, both derived from embryos cultured with 10% estrous cow serum, demonstrated significant fetal overgrowth. The increase in body weight relative to controls was similar for both groups (+22%, P < 0.001, and +19%, P < 0.001, respectively), but further analyses clearly separated the NT phenotype from the IVF phenotype. The NT-fetuses were characterized by a shorter crown-rump length but larger thorax circumference, which consequently produced a significantly reduced fetus length-to-thorax circumference ratio in comparison with IVF-fetuses and controls (-9% each, P < 0.0001). Absolute liver weight was significantly increased in NT- and IVF-fetuses (+62%, P < 0.0001, and +20%, P < 0.0001, respectively), but relative liver weight was increased only in NT-fetuses (+30%, P < 0.0001). Heart (P< 0.0001) and kidney (P = 0.0003) mass also showed disproportionate organomegaly in NT-fetuses only, but relative lung weight (NT, P = 0.263; IVF, P = 0.317) was not affected by either embryo technique. Transcript abundance for IGF2 and IGF2R genes were strongly correlated in muscle (r = 0.835, P < 0.0001), liver (r = 0.922, P < 0.0001), and lung (r = 0.772, P < 0.0001). The IGF2 and IGF2R transcript levels in muscle tissue from NT-fetuses were markedly reduced in comparison with both IVF-fetuses (-31%, P < 0.0001, and -41%, P < 0.0001, respectively) and controls (-31%, P < 0.0001, and -41%, P < 0.0001, respectively). In liver tissue, however, transcript levels for NT-fetuses were similar to those of controls, and IVF-fetuses showed markedly elevated, albeit non-significant, IGF2 (+86%, P = 0.0591) and IGF2R (+54%, P = 0.1305) mRNA levels relative to controls. Our data demonstrate that seemingly similar syndromes caused by NT or IVF procedures can be clearly partitioned with respect to phenotype and IGF2/IGF2R expression.
262 ASSESSMENT OF HSP70-1 TRANSCRIPTION LEVELS IN IMMATURE OOCYTES FROM BOS TAURUS AND BOS INDICUS COWS RAISED IN A TROPICAL CLIMATE
Authors: Camargo L. S. A. , M Viana J. H. , Serapião R.V. , Guimarães M. F. M. , Sá W. F. , Ferreira A. M.
Published: December 2006

Heat stress is one of the main causes of low conception rate in Bos taurus cows in a tropical climate. On the other hand, in this environment, oocytes from Bos indicus show greater developmental capacity after in vitro fertilization than those from Bos taurus, suggesting an adaptation to the hot climate. Heat shock proteins (HSP) are chaperones that promote protection against heat damage, and their transcription is associated to stress. The aim of this study was to evaluate the expression of HSP70-1 gene (Genbank NM174550), a member of HSP family, in oocytes from Bos taurus (Holstein) and Bos indicus (Gyr) cows raised in the tropical climate located at 21�35′′S latitude, 43�51′′W longitude, and 435 m altitude. Cumulus–oocyte complexes were recovered by oocyte pickup from mature non-lactating Holstein (n = 4) and Gyr (n = 4) donor cows during the hot season. Cumulus cells of viable oocytes were removed by vortexing in TALP-HEPES plus BSA, and pools (3 for each breed) with 12 immature oocytes were rapidly frozen in liquid nitrogen and subsequently thawed for RNA extraction. Total RNA extraction was performed using Rneasy� Micro kit (Qiagen, Valencia, CA, USA), and first strands were synthesized using SuperscriptTM III First Strand Synthesis kit (Invitrogen, Chicago, IL, USA). Relative quantification was performed in duplicate using real-time PCR (ABI Prism� 7000; Applied Biosystems, Foster City, CA, USA); reactions consisted of a mixture of iTaqTM SYBR� Green Supermix with ROX (Bio-Rad, Waltham, MA, USA) and cDNA equivalent to 1.2 oocytes and gene specific primers. Expression of the GAPDH gene was used as endogenous reference. Calculations of relative quantification were performed by the comparative Ct method, using the lowest value found in Bos indicus oocytes as calibrator; values (mean � SE) are shown as n-fold difference relative to the calibrator. Statistical comparison between breeds was performed by analysis of variance. Oocytes from Holstein cows showed a higher level (P < 0.05) of HSP70-1 expression (1.82 � 0.22) than oocytes recovered from Gyr cows (1.12 � 0.11). Previous study reported that oocytes from Gyr cows in a tropical climate showed a higher blastocyst rate after in vitro fertilization than Holstein oocytes (Camargo et al. 2006 Reprod. Fertil. Dev. 18, 243 abst). The lower level of HSP70-1 in Gyr oocytes suggests that they were less subject to stress than the Holstein ones, which may reflect their capacity to develop after fertilization. This effect may be, at least in part, due to the ability of Bos indicus cows to regulate body temperature in a hot environment, causing less stress on oocytes.
Financial support was provided by FAPEMIG, MG, Brazil, and CNPq, DF, Brazil. Thanks to Agrogen�tica, Vi�osa, Brazil, for the real-time PCR machine.