bluuebunny/crossref_metadata_2025 huggingface dataset:
156 PRETREATMENT WITH THE ANTIOXIDANT EPIGALLOCATECHIN GALLATE (EGCG) COUNTERACTS THE NEGATIVE EFFECTS OF MATERNAL HYPERTHERMIA ON EMBRYONIC DEVELOPMENT IN MICE
Authors: Aroyo A. , Yavin S. , Roth Z. , Arav A.
Published: December 2006

Hyperthermia-induced oxidative stress is one of the suggested mechanisms underlying the loss of developmental competence in heat-stressed embryos. The objective of the present study was to determine whether pretreatment with the antioxidant epigallocatechin gallate (EGCG) would counteract the negative effects of maternal hyperthermia on oocyte competence and improve subsequent embryonic development. Exp. 1 examined the effect of pretreatment with EGCG on ex vivo embryonic development under normal culture conditions (KSOM, 37°C, 5% CO2, 95% RH in air). Female mice (CB6F1) were synchronized (PMSG + hCG) and injected with 0.4 mL EGCG (100 mg/kg body weight) or with saline. Both EGCG- and saline-treated females were paired with stud males overnight. Mated mice were sacrificed and putative zygotes recovered and cultured in vitro. Cleavage and blastocyst formation rates were recorded on Days 1 and 5 post-fertilization, respectively. The percentage of putative zygotes that cleaved into the two-cell stage did not differ between the groups; however, blastocyst formation rate was higher (P < 0.05) in the EGCG group (85 ± 2%) than in the saline group (75 ± 2%). In Exp. 2 (a 2 × 2 factorial study), both EGCG- and saline-treated mice were exposed to normo-thermal conditions (NT; 22°C, 45% RH) or heat stress (HS; 40°C, 70% RH) for 1.5 to 2 h, the latter to induce a rise of 2°C in body temperature. Synchronized mice were paired with stud males overnight and mated mice were then sacrificed. Putative zygotes were recovered and cultured in vitro as described above. The number of putative zygotes recovered, cleavage and blastocyst formation rates and percentage of hatched blastocysts are presented in Table 1. Blastocyst formation rate was higher (P < 0.05) in the HS-EGCG group than in the HS-saline group. In addition, HS-EGCG embryos exhibited developmental competence similar to that of embryos from both NT groups. In summary, pretreatment with EGCG improved developmental competence under the described culture conditions. Furthermore, pretreatment with the antioxidant EGCG counteracted the negative effects of maternal hyperthermia. Further studies are warranted to evaluate the effect of pretreatment with EGCG on embryo quality.

Table 1.
Effect of pretreatment with EGCG on developmental competence of heat stressed oocytes
162 IMPROVED EMBRYO SURVIVAL AND QUALITY WITH EMCARE II
Authors: Harvey A. , Lane M. , Thompson J.
Published: December 2006

Collection of embryos exposes them to a number of stresses, including light, air, and changes in temperature. Improvement of holding media to reduce the impact of handling stresses on the embryo during in vivo collection and transfer is therefore beneficial to ensure maintenance of viability following transfer. The aim of this study was to compare the effect of holding IVP-derived blastocysts at 25°C in Emcare I (ECMI, Emcare, Dallas, TX, USA) with those held in Emcare II (ECMII), a proprietry formulation designed to reduce in vitro-induced stress. In vitro-produced bovine embryos were generated using standard protocols. Blastocysts were randomly allocated to either ECMI or ECMII (ICPBio, Aukland, New Zealand) on Day 7 and were held at 25°C for a period of 24 h, after which they were cultured in Cook Bovine Blast (Cook Australia, Brisbane, Australia) supplemented with 10% fetal calf serum for 48 h. At 24 and 48 h, embryos were scored for hatching, and a cohort removed for TUNEL staining at each time point. Differences were analyzed by Student's t-test. At both 24- and 48-h culture, hatching rates tended to be higher for embryos held in ECMII than in ECMI (Table 1). The level of apoptosis at 48 h was reduced in blastocysts held in ECMII (P = 0.06). Moreover, the total cell number of hatched blastocysts at 48 h was significantly increased (1.5-fold) in those held in ECMII (P = 0.01). Results suggest that the formulation of ECMII improves the ability of IVP bovine blastocysts to re-expand and hatch following an imposed stress (25°C for 24 h). Furthermore, ECMII improves overall embryo quality through a reduction in the percentage of cells undergoing apoptosis as well as through increased cell numbers, evident 48 h following cessation of the stress. We suggest that Emcare II reduces the impact of (or increases the embryo's tolerance to and recovery from) an imposed stress, which, although severe in the present study, may provide improved outcomes following embryo transfer in field situations.

Table 1.
Hatching and apoptosis of blastocysts held at 25°C for 24 h in Emcare I or Emcare II

This work was supported with funding by ICPBio (NZ).
163 SUCROSE ADDITION AT EARLY CULTURE STAGE IMPROVES DEVELOPMENT OF PRE-IMPLANTATION PORCINE NT AND IVF EMBRYOS
Authors: Hwang I.-S. , Seo J.-S. , Park H.-S. , Kim S.-W. , Kim D.-H. , Yang B.-C. , Yang B.-S. , Cheong H.-T. , Im G.-S.
Published: December 2006

Apoptosis is a form of cell death leading to fragmentation of the DNA, shrinkage of the cytoplasm, membrane changes, and cell death without lysis or damage to neighboring cells. It might contribute to the low developmental rate of in vitro-produced (IVP) embryos, but apoptosis in porcine embryos is still unclear. This study investigated the effect of sucrose in the culture medium on the development of porcine NT and IVP embryos. Oocytes were aspirated from the follicles in ovaries collected from a local abattoir, and then matured in TCM-199 for 40-44 h. Fresh semen was diluted and equilibrated at 16�C. A final concentration of motile spermatozoa was adjusted to 5 � 105 cells/mL in fertilization medium. Fetal fibroblast cells were prepared from a 35-day-old porcine fetus and used as donor cells. Embryos were cultured in PZM-3 supplemented with 0.05 M sucrose for 2 days, and then cultured in the PZM-3 without sucrose for 4 days at 38.5�C under 5% CO2 in air. Apoptotic cell death was analyzed by using a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. All data were subjected to a generalized linear model procedure (PROC-GLM) of the statistical analysis system (SAS; SAS Institute, Inc., Cary, NC, USA). NT and IVF embryos cultured in the medium supplemented with sucrose showed a significantly higher blastocyst formation rate than those cultured with no addition (28.8 vs. 20% and 32.3 vs. 17.9%; P < 0.05, respectively). For apoptosis, both NT and IVF embryos cultured in the medium with sucrose showed significantly lower frequency of apoptosis compared to embryos cultured in the medium without sucrose (3.4 vs. 6.3% and 0.6 vs. 1.8%; P < 0.05, respectively). Finally, the number of nuclei in NT blastocysts cultured in the medium with sucrose was higher than that of NT blastocysts cultured without sucrose (30.8 vs. 25.5; P < 0.05, respectively). However, the number of nuclei in the IVF blastocysts was not significantly different between groups. These results indicate that sucrose addition may increase the development of porcine NT and IVF embryos to the blastocyst stage and decrease the rate of apoptotic cells.
165 EFFECTS OF GLUCOSE CONCENTRATIONS ON IN VITRO DEVELOPMENT AND THE INTRACELLULAR OXIDATIVE STATE OF IN VITRO-PRODUCED PORCINE EMBRYOS
Authors: Karja N. W. K. , Kikuchi K. , Fahrudin M. , Manabu O. , Somfai T. , Noguchi J. , Kaneko H. , Nagai T.
Published: December 2006

The present study was conducted to elucidate the effects of glucose addition during Days 0 to 2 (the day of in vitro fertilization was defined as Day 0) on the developmental competence, intracellular reactive oxygen species (ROS) level, and glutathione (GSH) concentration of in vitro-produced pig embryos. Zygotes were obtained as reported previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033-1041), and cultured in NCSU-37 supplemented with 1.5, 3.5, 5.5, 10, and 20 mM glucose (glucose groups) or in NCSU-37 supplemented with 0.17 mM pyruvate and 2.73 mM lactate (Pyr/Lac group) from Days 0 to 2. Subsequently, embryos in all groups were then cultured in NCSU-37 added with 5.5 mM glucose until Day 6. Data were analyzed by ANOVA and are presented as percentage for blastocyst rate and as mean � SEM for blastocyst cell number. The blastocyst rates and blastocyst cell numbers in glucose groups were significantly lower compared to those in the Pyr/Lac group (blastocyst rate: 5.2, 13.8, 12.6, 16.3, and 13.5%, respectively, vs. 26.3%); blastocyst cell number: 41.8 � 3.2, 41.6 � 2.3, 42.2 � 3.2, 43.0 � 3.3, and 39.2 � 2.8, respectively, vs. 52.7 � 4.1). The ROS levels of Day 1 embryos were significantly higher when they were exposed to glucose, whereas the ROS levels of Day 2 embryos were higher only in the 1.5 mM and 3.5 mM glucose groups, compared to levels in embryos in Pyr/Lac group. There were no differences in the GSH concentrations of Day 1 embryos among the glucose groups and the Pyr/Lac group. Of Day 2 embryos, the GSH concentration was significantly lower only in 1.5 mM glucose group, compared to that in embryos in the Pyr/Lac group. These results indicate that (1) the presence of glucose during the first 2 days of culture causes a decrease in the development of in vitro-produced embryos to the blastocyst stage, which might be related to the rise in ROS generation in Day 1 embryos; and (2) except for the 1.5 mM glucose group, the ability of Day 2 embryos in glucose groups to maintain GSH concentration at levels needed for their development might provide them with preferable intracellular conditions for the development to the blastocyst stage.
166 EFFECTS OF LEPTIN AND IGF-1 ON PRE-IMPLANTATION DEVELOPMENT, DNA FRAGMENTATION, AND GENE EXPRESSION OF BOVINE EMBRYOS CULTURED IN VITRO
Authors: Kaya A. , Sagirkaya H. , Misirlioglu M. , Gumen A. , Memili E. , Parrish J. J.
Published: December 2006

Adequate regulatory proteins, growth factors, and hormones in in vitro embryo culture systems are important for improving the quality of embryos to a level similar to that in vivo conditions. The objective of this study was to define the effects of leptin, insulin-like growth factor-1 (IGF-1), and their combination on embryonic development, apoptosis, and expression profiles of a panel of developmentally important genes. Presumptive zygotes (16–18 h post-insemination) were randomly assigned and cultured in control (no supplementation), 5 ng/mL leptin (Group I), 100 ng/mL IGF-1 (Group II), and 5 ng/mL leptin and 100 ng/mL IGF-1 (Group III), all supplemented with 10% FCS on Day 4. On Day 8, the embryos reaching blastocyst stage were randomly either fixed for determination of DNA-fragmented nuclei by using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) or frozen for real-time relative quantitative RT-PCR analysis. The RT-PCR was performed to assess gene transcripts of glucose transporter-1 (Glut-1), heat shock protein 70.1 (Hsp70.1), interferon tau (IF-tau), insulin-like growth factor II receptor (IGF-IIr), desmosomal glycoprotein desmocollin III (DcIII), and DNA methyltransferase 3a (Dnmt3a). A total of 349, 322, 347, and 360 zygotes were used for the control group and Groups I, II, and III, respectively. Data were analyzed with a randomized complete block design and arcsine square root transformation of the dependent variables consisting of four treatments and six replicates. Cleavage rates were 79.5, 84.2, 87.3, and 82.4% for the control group and Groups I, II, and III, respectively, and only Group II was different from the control (P < 0.05). The percentages of embryos developed beyond the 8–16 cell stage were 44.2, 48.2, 49.0, and 50.7 for the control group and Groups I, II, and III, respectively, and Group III was different from the control (P < 0.05). Percentages of blastocyst development were 26.7, 29.6, 31.5, and 29.8, and the mean blastocyst cell numbers were 96.6, 98.6, 104.4, and 104.1 for the control group and Groups I, II, and III, respectively. The percentage of nuclei with fragmented DNA were 4.2, 3.3, 2.5, and 1.9 for the control group and Groups I, II, and III, respectively. Addition of IGF-1 and/or combination with leptin (Groups II and III) decreased the number of nuclei with fragmented DNA (P < 0.01) as compared to the control group. Although the expression of Glut1, DcIII, and Igf2r did not change among the groups, IF-tau and Dnmt3a were down-regulated in Group II. Hsp70 and IF-tau were up regulated in Group III. Results indicate that addition of IGF-I in culture media improved the cleavage rate; combination with leptin also improved the development rates to 8–16-cell-stage embryos, decreased the TUNEL-positive nuclei, and altered expression of some of the developmentally important genes.
170 POST-THAW VIABILITY OF IN VITRO-PRODUCED BOVINE EMBRYOS CULTURED EITHER IN HOST CAPRINE REPRODUCTIVE TRACTS OR IN VITRO
Authors: Menges S. , Bormann C. , Stroud B. , Kraemer D. , Westhusin M. , Long C.
Published: December 2006

In vitro culture of bovine embryos is usually associated with poor pregnancy rate following cryopreservation. The objective of this study was to compare the post-thaw viability of in vitro-produced bovine zygotes, cultured in vitro or in the reproductive tract of a host goat. Cumulus-oocyte complexes were matured in vitro, and in vitro fertilization was carried out with frozen-thawed semen as per standard laboratory procedures. At 18-20 h post-fertilization, zygotes were stripped of remaining cumulus cells and randomly separated into culture treatments. In three replicates, a total of 606 embryos were surgically transferred 12 to 24 h post-ovulation to the oviducts of an estrous-synchronized goat (VIVO) and 550 embryos were cultured in G1.3 for 72 h and then moved to G2.3 medium for 96 h and in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 (IVC). On Day 7, embryos were flushed from the excised tract with a 69.5% recovery rate or removed from culture. Embryos were classified according to IETS criteria with grades and stages recorded. All data were analyzed using the one-way analysis of variance and means were compared using Student's t-test. No differences were seen in the percentage of freezable quality embryos per total recovered between the two groups (34.3% vs. 32.3% for IVC and VIVO, respectively). However, there was a significant difference in the pre-freezing stage between the two culture groups (Stage 5.5 � 0.22 vs. Stage 4.8 � 0.26 for IVC and VIVO, respectively; P < 0.05), but no difference in the quality grade. All embryos greater than Stage 4, Grade 2 were frozen in groups of 5-10 in ethylene glycol with sucrose (Vigro Ethylene Glycol Freeze Plus; Bioniche Animal Health, Belleville, Ontario, Canada) in 0.25-mL straws. After thawing, embryo groups were washed, rehydrated, and incubated in G2.3 as above. Morphology was assessed by assigning grade and stage objectively at 24 h and 48 h post-thaw. Post-thaw viability in vitro was not different between groups (73.4% vs. 72.7% for IVC and VIVO, respectively). The average changes in morphology post-thaw from pre-freezing to 24 h and from 24 h to 48 h within each freezing group were determined. There was no significant difference in the mean change in stage (0.67 � 0.15 vs. 0.82 � 0.17 at 24 h and 0.31 � 0.09 vs. 0.37 � 0.10 at 48 h for IVC and VIVO, respectively) or grade (0.60 � 0.15 vs. 0.41 � 0.17 at 24 h and 0.03 � 0.06 vs. 0.14 � 0.07 at 48 h for IVC and VIVO, respectively) at either observation point. These results suggest that culture of in vitro-fertilized bovine embryos in the caprine reproductive tract did not alter post-thaw development or improve post thaw viability compared to in vitro cultured controls. However, morphological evaluation is too subjective to successfully predict pregnancy rate after transfer; therefore, further study is needed to determine if there are differences in pregnancy rates between these culture methods.
179 EFFECTS OF AN ENDOCANNABINOID, ANANDAMIDE, ON BOVINE BLASTOCYST DEVELOPMENT IN VITRO
Authors: Turco M. Y. , Matsukawa K. , Loi P. , Ptak G.
Published: December 2006

Cannabinoids cause many adverse effects on reproductive functions including fetal loss and pregnancy failure (Paria et al. 1995 PNAS 92, 9460–9464). N-arachidonylethanolamine (anandamide), an endogenous cannabinomimetic lipid derivative, binds with high affinity to brain type and spleen type cannabinoid receptors (CB1-R and CB2-R) and mimics most of the effects of Δ9-tetrahydrocannabinol [(−)THC], a psychoactive derivative of marijuana. In this study, we investigated the effects of anandamide on ovine blastocyst development in vitro. In vitro-matured oocytes were chemically activated and cultured to the blastocyst stage in our standard media (Ptak et al. 1999 Biol. Reprod. 61, 1568–1574). The development rate of blastocysts was 41%. Day 7 blastocysts were exposed to 28 nM anandamide with or without 20 nM SR141716A (an antagonist of CB1-R) for 48 h. In Experiment 1, we examined the CB1-R protein localization on blastocysts by anti CB1-R antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). In Experiment 2, to investigate the possible effect of anandamide on blastocyst development, we used the cell viability assay by propidium iodide, a cell proliferation assay (5-bromo-1′-deoxyuridine incorporation), and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (Qbiogene, Inc., Carlsbad, CA, USA). Data were analyzed by one-way ANOVA. CB1-R signals were detected on ovine blastocysts, and these signals were effectively improved in the anandamide co-cultured group. The results of Experiment 2 are summarized in Table 1. Our results demonstrate that CB1-R was expressed in the ovine blastocyst, and anandamide exerts negative effects on in vitro blastocyst development by inhibiting cell proliferation and increasing apoptotic rate, but not cell viability. Furthermore, SR141716A can effectively block the negative effects of anandamide.

Table 1.
Effect of anandamide (AEA) on sheep blastocyst development assessed by viability, cell proliferation, and TUNEL staining assays
180 DETECTION OF CASPASE-3 AND -7 IN NORMAL AND SLOW DEVELOPING BOVINE IN VITRO EMBRYOS
Authors: Vandaele L. , Mateusen B. , Maes D. , Van Soom A.
Published: December 2006

The incidence of apoptosis is an important indicator of embryo quality that can be measured by detection of active caspase-3 and -7 at various stages of in vitro development. The aim of the present study was to investigate the expression of these apoptotic markers in bovine in vitro embryos at different developmental stages using a fluorescent staining method. Additionally, expression of both caspases was compared between normal and slow-developing embryos. A total of 2640 immature bovine oocytes (four replicates) were matured and fertilized in vitro. Presumed zygotes (n = 2400) were denuded 24 h post-insemination (hpi) after fertilization and cultured in 50-μL droplets of modified SOF medium with 10% fetal calf serum at 39.0°C in 5% CO2, 5% O2 and 90% N2. Cleavage rate was determined at 45 hpi for all embryos, except 400 that were used as positive controls for the caspase staining. At different time points, embryos were allocated to the normal developing group if they reached the 5–8-cell stage at 45 hpi; the 9–16-cell stage at 80 hpi, the morula stage at 117 hpi and the expanded blastocyst stage or beyond at 168 hpi. Embryos that lagged one cell cycle behind at each time point were designated as slow-developing embryos. All selected embryos were stained using CaspaTag Pan-Caspase In Situ Assay kit, Fluorescein® (Chemicon International, Temecula, CA, USA). Positive controls were incubated in 0.5 μM staurosporine (Sigma, Belgium) for 24 h. The apoptotic cell ratio (ACR) was determined for each embryo by means of confocal laser scanning microscopy (ACR = number caspase-positive cells/total cell number × 100). Control embryos (n = 400) were cultured undisturbed until 168 hpi and evaluated for blastocyst yield. Mixed model analyses of variance with group as fixed factor and replicate as random factor were used to evaluate the ACR at 45, 80, 117, and 168 hpi, respectively. The mean cleavage rates at 45 hpi and the blastocyst yield at 168 hpi were 60.5% and 26.9%, respectively. The ACR over all time points and at each individual time point was significantly lower in normal-developing embryos compared with slow-developing embryos, as determined by caspase staining (P < 0.01) (Table 1). In conclusion, this study demonstrated that slow-developing embryos express a higher level of apoptosis. Further research is necessary to investigate the cause of the higher ACR in slow developing embryos, which may be the result of altered gene expression or environmental influences.

Table 1.
Apoptotic cell ratio (ACR; mean % ± SD) at four time points in normal and slow-developing embryos

This study was supported by a grant of Research Foundation – Flanders (aspirant 1.1.084.04N00).
181 MOUSE TRIPLETS DEVELOPED FROM SINGLE BLASTOMERES OF AN 8-CELL-STAGE EMBRYO SUPPORTED WITH TETRAPLOID EMBRYOS
Authors: Bogdan C. V. , Catunda A. P. , Bodo S. , Ilie D. , Kovacs A. , Gocza E.
Published: December 2006

Chimera production using altered ES cells became a key tool for generating transgenic mice. However, chimeras are more than just a tool for making mouse mutants; they play a crucial role in analyzing the biological effects of genetic changes. Chimeras can be made by combining two whole 8-cell embryos or by combining subsets of blastomeres of two cleavage stage embryos. Because, at these stages, the early embryonic cells are not yet restricted in their lineage potency, they are equally capable of contributing to the inner cell mass or the trophectoderm. Pluripotency of single blastomeres of the 4-cell and 8-cell mouse embryo has been proved indirectly by aggregating them with carrier blastomers of a different genotype, giving rise to chimeric blastocyst (Tarkowski et al. 1967 J. Embryol. Exp. Morphol. 18, 155-180). In our study we wanted to demonstrate that a single blastomere of an 8-cell stage embryo, supported with tetraploid embryos at the 4-cell stage, is capable of developing into a healthy animal. When tetraploid embryos are used to make chimeras together with diploid cells, tetraploid cells rarely contribute to the embryo itself, but contribute mainly to the primitive endoderm and the trophectoderm, so in this case the newborns will be derived only from the diploid blastomers used (Nagy et al. 1990 Development 110, 815-821). We produced chimeras by aggregating a single blastomere [(2n)(1-cell)] derived by combining sexed, GFP-expressing, diploid 8-cell stage embryo (Hadjantonakis et al. 1998 Nat. Genet. 19, 220-222) with either a sexed diploid 7-cell embryo [(2n)(7-cells)] (one of the 8-cells was taken for sexing) or a non-sexed tetraploid embryo [(4n)(4-cells)]. The aggregates were cultured in vitro and transferred as blastocysts to the uteruses of pseudo-pregnant females. Fetuses were removed by Caesarian section and raised by lactating foster mothers. From the transferred 84 [XY(2n)(1-cell)]/[XX(2n)(7-cells)] aggregates we obtained 12 (14.3%) newborns, 11 (91.7%) males and one (8.3%) female. From the transferred 27 [XY(2n)(1-cell)]/[(4n)(4-cells)] aggregates, where one XY blastomere was combined with a tetraploid embryo, we obtained 7 (25.9%) male newborns: 1 triplet and 2 pairs of twins. We also transferred four [XX(2n)(1-cell)]/[(4n)(4-cells)] chimera embryos, where an XX-blastomere was aggregated with a tetraploid embryo; we obtained a set of living female triplets (75%) from this aggregate. We demonstrated that a single blastomere of the 8-cell-stage embryo is capable of developing into a living newborn. We obtained identical pups from three blastomeres isolated from the same embryo (triplets). This way we produced single blastomere clones and could control the sex of the new generation.
This research was supported by grants from OTKA T037582 and RO-1/2002 Intergovernmental S&T cooperation program.
182 FAST STAINING METHODS FOR DIFFERENTIAL STAINING OF INNER CELL MASS AND TROPHECTODERM CELLS OF MAMMALIAN BLASTOCYSTS
Authors: Kim S. W. , Park J.-K. , Han J.-H. , Park C. G. , Chang W.-K.
Published: December 2006

The present study was undertaken to develop a simple differential staining method for inner cell mass (ICM) and trophectoderm (TE) cells of mammalian blastocysts using the permeabilizing agent, saponin, without species-specific antibodies and complements. The nuclei of whole embryos were pre-stained to green by 5 �M SYTO 13 for 10 min. After washing, the green color of TE was turned to red by exposure to 100 �g/mL propidium iodide and 50 to 100 �g/mL saponin solution for 5 to 10 min. To confirm the exactness of staining patterns, the fluorescent nuclei of ICM and TE from mouse, pig, and bovine blastocysts were compared with 3D location by confocal microscopy. By the saponin mixture treatment method, in vitro-cultured mouse, pig, and bovine blastocysts were shown to have an ICM:TE ratio of 1:2.5, 1:4.5, and 1:3.6, with an average total cell number of 78 � 14 (n = 45), 65 � 18 (n = 49), and 150 � 20 (n = 45), respectively. Although a few TE cells were stained to a yellowish-green color, the successful protection of the green color of ICM depended on the exposure time of blastocysts to the saponin mixture. The total time lapse of the procedure did not exceed 1 h. These results indicate that saponin could be used as a practical substitute for special antibodies and complements. So this differential staining for examining the ICM:TE ratio and the total cell count of mammalian blastocysts would be a fast and reliable method.
183 DEVELOPMENT OF PORCINE EMBRYOS MICROINJECTED WITH PORCINE EMBRYONIC GERM CELLS
Authors: Park C.-H. , Lee S.-G. , Choi D.-H. , Kim M.-G. , Lee C. K.
Published: December 2006

Embryonic germ (EG) cells, derived from primordial germ cells in the developing fetus, are similar to embryonic stem (ES) cells in terms of expression pattern of undifferentiated markers and their ability to colonize both the somatic and the germ cell lines following injection into a host blastocyst, which has been proven in mouse. Several studies using porcine EG cells have shown that it is possible to produce somatic chimeras after blastocyst injection. However, not only was the degree of reported chimerism low, but also there has been no report about the fate of injected EG cells in porcine blastocysts. This study was designed to observe the distribution pattern of porcine EG cells in chimeric blastocyst after injection into cleavage-stage porcine embryos. To ascertain development of microinjected porcine embryos with EG cells, 10 to 15 EG cells were injected into cleavage stage of in vitro fertilized embryos and cultured up to blastocyst. Also, porcine EG cells were labeled with DiO (Invitrogen, Carlsbad, CA) on the cell membrane or transfected with green fluorescent protein gene to observe whether the EG cells injected in the host embryo would incorporate into the inner cell mass (ICM) or trophectoderm (TE). Chimeric embryos were produced and allowed to develop into blastocysts to investigate the injected EG cells would come to lie in ICM and/or TE of the blastocyst, by scoring their position. In result, developmental rate was similar in all treatments. In all treatments, EG cells were mainly allocated in both ICM and TE of the chimeric blastocysts. These results suggest that examining the allocation pattern of injected EG cells, maintained pluripotency in vitro, could provide clues of differentiation process in vivo. Furthermore, to enhance the allocation of EG cells into the embryonic lineage, it would be required to optimize the culture condition for EG cells as well as embryos. Further experiment are needed to determine whether the injected EG cells could maintain their properties throughout the environment in the embryonic development in vitro.

Table 1.
Distribution of the porcine EG cells microinjected into cleavage-stage embryos
188 EFFECT OF TRANSFER OF FROZEN-THAWED IVF EMBRYOS ON PREGNANCY IN REPEAT-BREEDER INSEMINATED OR NON-INSEMINATED HOLSTEIN CATTLE
Authors: Dochi O. , Tanisawa M. , Goda S. , Koyama H.
Published: December 2006

Repeat-breeding is one of the important factors that affect dairy management. The objective of this study was to investigate the effect of transfer of frozen–thawed IVF embryos on pregnancy in repeat-breeder Holstein cattle. Cumulus–oocyte complexes (COCs) were collected by aspiration of 2–1-mm follicles from ovaries obtained at a local abattoir. COCs were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 mg/mL of FSH at 38.5°C under a 5% CO2 atmosphere in air. Matured oocytes were inseminated with spermatozoa of 5 × 106/mL in BO solution (Brackett and Oliphant 1975 Biol. Reprod. 12, 260–274) containing 10 mM hypotaurine and 4 units/mL heparin. After 18 h of gamete co-culture, presumptive zygotes were cultured in CR1aa (Rosenkrans et al. 1991 Theriogenology 35, 266) supplemented with 5% CS for 8 days at 38.5°C under 5% CO2, 5% O2, 90% N2 atmosphere in air. After in vitro fertilization, Day 7 and Day 8 blastocysts were frozen in 1.5 M ethylene glycol (EG) in Dulbecco's PBS (DPBS) supplemented with 0.1 M sucrose and 20% CS. Embryos were transferred into a freezing medium, loaded into 0.25-mL straws, and allowed to stand for 15–20 min for equilibration. The straws were then plunged into a −7°C methanol bath of a programmable freezer for 1 min, seeded at −7°C, maintained at −7°C for 15 min, cooled to −30°C at the rate of −0.3°C/min, and then plunged into liquid nitrogen. Recipient animals (43 heifers, 131 cows) included those that did not conceive after being artificially inseminated (AI) 3 to 15 times. The frozen–thawed IVF embryos were directly transferred to the recipient animals 7 days after estrus or AI. Pregnancy rates were analyzed by chi-square test. The results are presented in Table 1. There were no significant differences in the pregnancy rates between treatments. However, a slightly higher pregnancy rate was achieved by embryo transfer after AI. These results suggest that embryo transfer may increase the pregnancy rate in repeat-breeder Holstein cattle.

Table 1.
Pregnancy rates after transfer of IVF frozen–thawed embryos in repeat-breeder Holstein cattle
195 DEVELOPMENT OF INNER CELL MASS AND FORMATION OF EMBRYOID BODIES ON A GELATIN-COATED DISH AND ON THE FEEDER LAYER IN BUFFALO (BUBALUS BUBALIS)
Authors: Chauhan M. S. , Verma V. , Manik R. S. , Palta P. , Singla S. K. , Goswami S. L.
Published: December 2006

Isolation and culture of embryo-derived cell lines have been reported in many mammals, however, there is not even a single report toward initiation of such work in buffalo (Bubalus bubalis). Therefore the present study was carried out to isolate the inner cell mass from in vitro-produced buffalo blastocysts and to grow this inner cell mass for formation of embryoid bodies in a gelatin-coated dish and on an homologous fetal fibroblast feeder layer. Immature buffalo oocytes were isolated from the slaughterhouse ovaries. In vitro production of blastocysts was carried as reported by Chauhan et al. (1999 J. Dairy Science 82, 918-926). A total of 26 buffalo blastocysts were produced in vitro. These blastocysts were transferred into a 100-mL drop of Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS) for further culturing. Thirteen blastocysts hatched on the next day of culture. The hatched mass was separated, suspended in Dulbecco's phosphate buffer saline (DPBS, without Ca++ and Mg++) containing 5% FBS + 0.25% trypsin and examined under the zoom stereomicroscope until disappearance of the trophectoderm cells. The remaining cells of seven blastocysts were cultured in DMEM supplemented with 20% FBS on 0.2% gelatin coated culture dish (group 1), and cells of six blastocysts in DMEM medium supplemented with 20% FBS were cultured on a mitomycin-c-treated (10 �L/mL) feeder layer (group 2), for 14 days. The isolated cells attached to the bottom of the dish in both the groups, spreading was noticed on Day 5 of the culture in group 1 and on Day 3 of culture in group 2. The attached cells were trypsinized using DPBS with 0.25% trypsin, isolated, and subcultured further. Attachment and spreading was noticed only in group 2 subcultured cells. The cellular integrity was homogeneous and the plasma membrane was clearly visible in group 2, but not in group 1. Less than 10% of the attached cells formed embryoid bodies in group 1, where as more than 30% attached cells in group 2 formed embryoid bodies; the latter expressed alkaline phosphatase activity and were blue after staining. These results indicate that the culturing of the inner cell mass on an homologous fetal fibroblast feeder layer is a better choice for production of embryonic stem cells in buffaloes.
196 EFFECT OF MITROMYCIN-C ON VIABILITY AND CELL PROLIFERATION OF DOMESTIC CAT AND MOUSE EMBRYONIC FIBROBLASTS
Authors: Gómez M. C. , Jenkins J. A. , López M. , Serrano M. A. , Dumas C. , Dresser B. L. , Pope C. E.
Published: December 2006

Nondomestic cat cloned embryos created by intergeneric nuclear transfer (ig-NT) have lower rate of blastocyst development than that observed in cloned embryos produced by inter-species NT. One promising technique for increasing the efficiency of ig-NT is to use embryonic stem cells (ESC) derived from the pluripotent inner cell mass (ICM) of pre-implantation blastocysts. Mouse embryonic fibroblasts (MEF) are commonly used as feeder cells to support the growth of human and mouse ESC, but they do not always maintain human ESC in an undifferentiated state and carry the risk of transmitting mouse retroviral virus and other pathogens. Thus, it is important to determine whether domestic cat embryonic fibroblasts (DCEF) are able to support cat ESC growth. Proliferation of MEF cells is inhibited before their use as feeder layers by exposure to gamma irradiation or Mitomycin-C. Therefore, in the present study, we determined what concentration of Mitomycin-C was required to inhibit proliferation of DCEF and MEF cells without affecting viability. Embryonic fibroblasts were generated from fetuses collected from a single pregnant domestic cat and mouse at 30 and 14 days of gestation, respectively. Tissue was minced and cultured in DMEM for 7 to 10 days before freezing and storage at −196°C. After thawed DCEF and MEF cells reached 80 to 100% confluence, proliferation was inhibited by exposure to 10, 30, or 40 µg of Mitomycin-C for 2.5 or 5 h. Cells were then washed and labeled with BrdU to measure DNA synthesis as a specific marker for replication. For cytotoxicity, cells were labeled with a dual fluorescent stain to evaluate membrane integrity and esterase activity. The amounts of DNA-labeled BrdU as well as calcein and ethidium homodimer-1 incorporated into the cells were quantified by flow cytometry (see Table 1). Proliferation of DCEF and MEF cells was affected similarly by both concentration and exposure time to Mitomycin-C, with the highest inhibition of DCEF cells occurring after treatment with 40 μg for 5 h (92% not replicating). Cell viability was influenced by both concentration and exposure time to Mitomycin-C, with the highest survival of DCEF cells occurring after exposure to 30 μg for 5 h (90%) and in MEF cells after exposure to 40 μg for 2.5 h (88% viable). In summary, these results indicate that cell proliferation of DCEF can be inhibited by Mitomycin-C, but a higher dosage and longer exposure time should be considered for use compared to the dosage commonly employed for inhibiting cell proliferation of MEF. High concentrations of Mitomycin-C did not significantly increase cytotoxicity of cells in either species.

Table 1.
198 REPROGRAMMING FIBROBLAST CELL CULTURES WITH EMBRYONIC STEM-CELL EXTRACTS
Authors: Kobolak J. , Rozsar H. , Fellinger E. , Dinnyes A.
Published: December 2006

Reprogramming differentiated cells could be a effective method to dedifferentiate and transdifferentiate cells and thereby to replace or treat tissues. The goal of our study was to examine the effectiveness of embryonic stem (ES) cell extract on reprogramming fibroblast cell cultures and to use these cells as nucleus donors in subsequent cloning experiments. Based on the method of Collas and Hakelien (2003 Trends Biotechnol. 21, 354-361), cell extracts were produced from ES cells after homogenization and sucrose gradient centrifugation. The ES cells were suspended in one volume of 0.3 M sucrose then one volume of 2 M sucrose was added, and cells were homogenized. Finally 5.6 volumes of deionized H2O were added to the suspension, and the nucleus compartments were separated with centrifugation (2000 rpm, 10 min; Janetzki K24 angle rotor). The supernatant was separated (cytosol fragment), the precipitate was rehomogenized in the same way (nuclei fraction), and both fractions were centrifuged (8000 rpm, 1 h, Janetzki K24 angle-rotor). The supernatants were ultracentrifuged (40 000 rpm, 30 min; Beckman 40 angle rotor) and used immediately or stored at -80�C. Fibroblast cells were cultured and treated in 24-well plates. Cell extracts were administered to culture media [DMEM + 10% fetal bovine serum (FBS)] directly. Cell numbers were calculated after MTT-assay (Roche, Budapors, Hungary). Cell divisions were monitored by BrDU-tests (Roche) and RT-PCRs (Cdk2 and p21; Invitrogen, Budapest, Hungary). Results were statistically analyzed with Student's t-test. In our experiments the crude cell fractions killed 98% (P < 0.005) of cells after 1-h treatment, thus the administration of protease inhibitors was necessary. The supplemented extracts were tested as to dosage and treatment time: 4-h treatment (P < 0.05) and 105 or 106 cells/mL (P < 0.05; cell number in 1 mL of starting material) were the most appropriate combination. When 5 mM CaCl2 was administered, the cell cycle-arresting effect was increased using either the nuclear (+24%, P < 0.05) or the cytosol extract (+30%, P < 0.05), but the effect of the nuclear extract was more intensive. When steptolysin O (0.1 �g, 5 min) treatment of fibroblast was used prior to addition of ES cell extracts, 1-h treatment and 104 cells/mL were the most effective combination, as higher concentration or longer treatment was toxic. Further experiments are needed to test the effect of treatment on nuclear transfer procedures.
This research was supported by MEXT-CT-1003-19582; Wellcome Trust (Grant No. 070246); OTKA T046171; and NKTH BIO-10017/2002.
20 PRODUCTION OF NUCLEAR TRANSFER EMBRYOS FROM PORCINE FETAL FIBROBLAST CELLS CARRYING A TETRACYCLINE-INDUCIBLE TRANSGENE
Authors: Ahn K. S. , Kwon M. , Koo B. C. , Won J. Y. , Heo S. Y. , Kim T. , Shim H.
Published: December 2006

Constitutive expression of A transgene often results in serious physiological disturbances in transgenic animals. For instance, systemic overexpression of human growth hormone in transgenic pigs has resulted in detrimental side effects in general health and reproductive performance. One of the solutions to such problem would be inducible expression of a transgene that may restrict production of foreign proteins from transgenic animals only when needed. In this study, a retrovirus vector was designed to express the green fluorescent protein (GFP) gene under the control of the tetracycline-inducible promoter. Transformation of porcine fetal fibroblast cells was achieved by infection of the cells with the vector and subsequent antibiotic selection. To induce transgene expression, transformed porcine fetal fibroblast cells were cultured in medium supplemented with doxycycline for 48 h. Induction of the GFP gene was verified by the emission of fluorescence from transformed cells. Nuclei of transformed cells with or without doxycycline treatment were transferred into enucleated oocytes, and the induction efficiency was analyzed by monitoring fluorescent emission during development of reconstituted embryos to the blastocyst stage. In addition, differences in the rates of blastocyst development between experimental groups were analyzed by Student's t-test. Blastocyst formation of nuclear transfer embryos using transformed cells with tetracycline-inducible retrovirus vector (12.0%, 128/1072) was not significantly different (P > 0.05) from that with non-inducible control vectors (13.7%, 41/300), suggesting that an introduction of tetracycline-inducible retrovirus vector was not particularly harmful to the development of nuclear transfer embryos. Also, the blastocyst development rate of nuclear transfer embryos after induction of transgene by doxycycline (12.1%, 99/815) was not significantly different (P > 0.05) from that of the non-induced counterparts (11.3%, 29/257), suggesting that the induction of transgene did not affect the development of transgenic clone embryos. In a majority of embryos, high expression of the GFP gene was observed in cloned embryos with transgene induction, whereas poor or no GFP expression was detected in non-induced controls. The results from this study suggest that tetracycline-inducible expression of transgenes in nuclear transfer embryos may be used for production of foreign proteins in transgenic animals in a more controlled manner than with conventional procedures. Further experiments on transfer of cloned embryos carrying such an inducible transgene to recipients may enable production of transgenic pigs with fewer side effects from unregulated expression of the transgene.
202 IN VITRO DIFFERENTIATION OF ADULT PORCINE ADIPOSE-DERIVED STEM CELLS AFTER LABELING WITH PKH26 AND FLOW CYTOMETRY
Authors: Mello M. , Lima A. , Malusky S. , Lane S. , Wheeler M.
Published: December 2006

The purpose of this study was to investigate the possible effects of the fluorescent dye PKH26 and flow cytometry on adult porcine adipose-derived stem cells (ADSCs) after exposing them to adipogenic and osteogenic differentiation conditions. Adipose tissue was isolated from swine (11 months of age) and digested with 0.075% collagenase at 37�C for 90 min. The digested adipose tissue was centrifuged at 200g for 10 min to obtain a cell pellet. The pellet was re-suspended with DMEM, and the ADSCs were plated onto 75 cm2 flasks (5000-10 000 cells per cm2) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% gentamicin. Passage 3 cells were labeled with fluorescent dye (PKH26 red fluorescent cell linker kit; Sigma Chemical, St. Louis, MO, USA) and sorted by flow cytometry. After labeling and sorting, the sorted and unsorted (control group) cells were replated and exposed to adipogenic (1 �M dexamethasone, 0.5 mM isobutylmethylxantine, 10 �M insulin and 200�M indomethacin) and osteogenic (0.1 �M dexamethasone, 10 mM �-glycerophosphate, and 50�M ascorbic acid) differentiation conditions when the cells were 90% confluent. Cells were evaluated based on morphology and specific staining properties. Adipogenic differentiation was confirmed by oil red O-positive staining of large lipid vacuoles, and osteogenic differentiation by Von Kossa staining 2 weeks after initiation of differentiation. The frequency of oil red O-positive colonies in both sorted and unsorted group was similar (15.0% vs. 13.2%, respectively). The number of osteogenic nodules, confirmed by the presence of calcium by Von Kossa staining, in the sorted and unsorted group was 17 and 184 per flask, respectively. In conclusion, this study demonstrates that adult porcine adipose-derived stem cells maintain their differentiation potential after labeling with fluorescent dye and sorting by flow cytometry. This should allow for more rapid evaluation of the differentiation potential of ADSCs in vitro.
This work was partially supported by the Council for Food and Agricultural Research (C-FAR) Sentinel Program, University of Illinois and CNPq, Brazil (M. Mello).
203 EFFECT OF OXYGEN TENSION AND SUBSTRATE ON GROWTH AND DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS
Authors: Ramírez M. A. , Pericuesta E. , Pérez-Crespo M. , Fernández-González R. , Moreira P. N. , Rizos D. , Pintado B. , Gutiérrez-Adán A.
Published: December 2006

Normally the majority of mammalian cells, including murine embryonic stem (mES) cells, are immersed in a low oxygen environment (hypoxia); however, mES are generally cultured in an atmosphere containing 21% O2 (normoxia). Such conditions may not be the most appropriate for mES propagation. We have tested the effects of hypoxia and culture on either feeder fibroblasts or gelatin substrate on mES cell growth and spontaneous differentiation. Two ES cell lines (R1 129/Sv from the laboratory of A. Nagy and MAR B6D2 F1 generated in our laboratory) were cultured under two different oxygen tensions (5 and 21%), and on two different substrates, 0.1% gelatin or murine embryonic fibroblasts (mEF). Cell cycle, cell proliferation, mRNA expression of pluripotency and differentiation markers, as well as spontaneous differentiation to cardiomyocytes, were monitored. For cell proliferation measurements, mES after 7 days of culture at the different conditions were labeled with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester, and cultured for up to three more days. Cells were then harvested for flow cytometry analysis every 24 h after labeling (Cell TraceTM CFSE Cell Proliferation Kit; Molecular Probes, Inc., Eugene, OR, USA). For cell cycle analysis, cells grown on mEF under the two different oxygen tensions were fixed after 10 days of culture, and then stained with propidium iodide/Triton-X-100 for flow cytometry analysis (Current Protocols in Cytometry, Chap. 7, 2001). The spontaneous differentiation of embryoid bodies [formed by ES cells in the absence of leukemia inhibitory factor (LIF)] to cardiomyocytes was also monitored. For mRNA expression of pluripotency (Nanog, Oct-3/4, Rex1, GENESIS, FGFR-4, TERF1, Cx43, and GLUT1) and differentiation markers (GATA4, GATA2, AFP, Msx-1, Brachyury, and Myf5), RT-PCR analysis was performed on mES cells from Day 0 to Day 10. Under hypoxia conditions, the proliferation of both types of mES cells was greater than under normoxia, independent of substrate used, and a higher number of apoptotic cells was detected. Moreover, only under normoxia conditions did mES cells lose the expression of pluripotency markers GENESIS and GLUT1. In addition, under lower oxygen tension, the rate of differentiation to beating cardiomyocytes was significantly lower on the feeder layer than that under normoxia (11.9% vs. 28.1%; P = 0.012). The feeder layer supported significantly higher cardiomyocyte formation when compared to 0.1% gelatin at 21% O2 (28.1% vs. 8.3%; P < 0.001). Our results show that normoxia may not be the most appropriate condition for mES cell propagation and that hypoxic culture may be necessary to maintain full pluripotency of mES cells.