bluuebunny/crossref_metadata_2025 huggingface dataset:
253 DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES MATURED IN SERUM-FREE MEDIUM UNDER LOW OXYGEN TENSION
Authors: Pereira M. M. , Costa F. Q. , Campos P. H. A. , Serapiao R. V. , Polisseni J. , Carvalho B. C. , Viana J. H. M. , Sa W. F. , Machado M. A. , Camargo L. S. A.
Published: December 2008

In vitro maturation (IVM) is a critical step in in vitro bovine embryo production. A number of factors can influence the IVM environment, such as media composition and protein supplementation. Serum and higher O2 tension have been shown to reduce embryo quality; however, little is known about the effects of serum and O2 tension during IVM on embryo quality and development. This study aimed to evaluate the effect of serum and O2 tension on IVM of bovine oocytes. Immature oocytes obtained from slaughterhouse ovaries were randomly distributed in 4 groups of IVM: G1 (n = 253), 0.1% polyvinyl alcohol (PVA) in air; G2 (n = 248), 10% inactivated estrous cow serum (ECS) in air; G3 (n = 270), 0.1% PVA under 5% O2; and G4 (n = 236), 10% ECS under 5% O2. In vitro maturation was performed with TCM-199 culture medium supplemented with 20 μg mL–1 FSH, under 5% CO2 at 38.5°C for 24 h. After maturation, oocytes were in vitro fertilized with 2.0 × 106 sperm mL–1 in Fert TALP medium, supplemented with heparin, for 20 h. Presumptive zygotes were denuded by vortexing and cultured in CR2aa medium with 2.5% fetal calf serum under 5% CO2 and 5% O2 at 38.5°C. Cleavage rate was evaluated 72 h postfertilization, and blastocyst rate and total cell number were evaluated 8 days postfertilization. Morphological classification of embryos was performed at Day 8 according to the International Embryo Transfer Society manual (1998). Cleavage, blastocyst, and grade 1 embryo rates were analyzed by chi-square, and total cell number was analyzed by ANOVA, with means compared by LSD. Results are presented as mean ± SEM. There was no difference (P > 0.05) in cleavage rates among G1, G2, and G4 (61.6, 65.3, and 57.6%, respectively), but cleavage rate was lower (P < 0.05) in G3 (52.5%). Blastocyst rates among G1, G3, and G4 (15.8, 17.7, and 20.3%, respectively) were similar (P > 0.05). However, blastocyst rate in G2 (25.0%) was higher (P < 0.05) than in G1 and G3, but was similar to G4 (P > 0.05). Total cell number was similar (P > 0.05) among G2 (194.1 ± 13.1), G3 (173.3 ± 9.0), and G4 (163.8 ± 8.7), but was lower (P < 0.05) in G1 (124.5 ± 11.4). The grade 1 embryo rate was lower (P < 0.05) in G1 (70.3%) than in G2 (89.5%), but was similar (P > 0.05) to G3 (77.0%) and G4 (83.9%). The results suggest that IVM with PVA in TCM-199 medium under 5% O2 can be performed without reducing embryo development and quality, when compared with ECS. On the other hand, oocyte developmental competence seems to be affected when IVM is performed with PVA under air conditions.

Financial support: CNPq, FAPEMIG.
260 ULTRARAPID NON-ELECTOPHORETIC PCR-SEXING OF BOVINE EMBRYOS
Authors: Bredbacka P. , Reinikainen E.
Published: December 2008

In recent years, many improvements have been made in bovine embryo sexing. The challenge has been to create a fast, yet simple and user-friendly assay that can be performed with inexpensive equipment. The latter characteristics are featured in the widely used nonelectrophoretic Ampli-Y™ PCR assay (Finnzymes Oy) in which tubes with male embryo biopsies result in pink fluorescence under UV light (Bredbacka et al. 1995 Theriogenology 44, 167–176). However, until now the post-biopsy assay time has remained at about 1.5 h, which is impractical under field conditions. The recent developments of polymerases, thermal cyclers, and reaction tubes have presented an opportunity to improve the speed of sex analysis. This study aimed at optimizing a faster protocol for nonelectrophoretic sexing by taking advantage of the new developments. The Ampli-Y kit was the starting point for the new assay. The polymerase was replaced by the extremely fast Phusion™ DNA Polymerase (Finnzymes), in which a unique dsDNA-binding domain is fused to a Pyrococcus-like proofreading polymerase. The oligonucleotide primers were redesigned to allow higher annealing temperature and, hence, faster thermal cycling. The reaction tubes were replaced with ultra-thin walled PCR tubes (UTW™, Finnzymes) allowing quicker sample thermal response. For thermal cycling, the MJ Research Minicycler was replaced with the 24-well Piko™ Thermal Cycler (Finnzymes) enabling faster ramp rates. Different PCR protocols were tested by adding 100 pg of male or female DNA into the reaction vessels. The running time of the resulting optimized PCR protocol (initial denaturation 30 s at 98°C, followed by 38 cycles of 0 s at 95°C and 30 s at 72°C) was 35 min. The optimized PCR protocol was tested with Day 7 bovine morulae and blastocysts produced by standard IVP technology. Each of the 24 embryos was split into four biopsies. Of each embryo, one biopsy was subjected to electrophoretic based sexing (Bredbacka and Peippo 1992 Agric. Sci. Finl. 2, 233–238), one was subjected to sexing by the standard Ampli-Y™ assay, and the remaining two biopsies were each subjected to the optimized protocol. Some single blastomeres that detached from the embryo during biopsying were also assayed using the new protocol. Biopsies and blastomeres were lysed in PCR buffer containing polymerase K for 4 min at 55°C followed by 2 min at 98°C. The embryos were diagnosed as 15 males and 9 females with all assays giving the same result for each embryo. Male samples tested with the optimized protocol had all clear signals easily distinguishable from the female samples. The male single blastomeres showed fluorescence levels equal to that of the male biopsies indicating high sensitivity of the new assay. In conclusion, the optimized sexing protocol offers a significant time saving. Following biopsy and a 6 to 7 min lysis step, the results are obtained in 35 (±1) min, about 1 h shorter than with the original Ampli-Y™ protocol.
270 PRETREATMENTS OF PORCINE SPERM BEFORE INTRACYTOPLASMIC SPERM INJECTION AFFECT QUANTITY OF PLCZETA;
Authors: Nakai M. , Ito J. , Sato K. , Noguchi J. , Kaneko H. , Kashiwazaki N. , Kikuchi K.
Published: December 2008

In pigs, the intracytoplasmic sperm injection (ICSI) procedure alone is insufficient to induce oocyte activation for embryonic development. Artificial activation can be accomplished, such as by electrical pulse-enhanced in vitro development to the blastocyst stage (Nakai et al. 2006). It is well known that the sperm factor (phospholipase Cζ; PLCζ) in spermatozoa, which triggers oocyte activation, is diffused into ooplasm when sperm fuse with oocytes. Our previous study showed that the activation rate of porcine oocytes injected with one sonicated sperm head was significantly lower than that of oocytes injected with a whole spermatozoon or with 3 sonicated sperm heads (Nakai et al. IETS 2007). These results suggest that the sonication treatment per se may affect the quantity of PLCζ in sperm. Furthermore, various pretreatments of sperm besides sonication have been conducted (e.g. removal of the sperm membrane) to increase the efficacy of ICSI. In this study, we investigated the effect of pretreatments (sonication, Triton X-100, and repeated cycles of freezing–thawing without cryoprotectant) on the quantity of PLCζ in porcine sperm. Cryopreserved-thawed boar-ejaculated sperm were used for 3 experimental groups: (1) sperm were sonicated for 10 s in pig-fertilization medium (pig-FM; Suzuki et al. 2002; Soni group), (2) freezing–thawing was repeated 3 times in pig-FM without cryoprotectant (3-F/T group), or (3) sperm were incubated in pig-FM supplemented with 0.1 or 1% Triton X-100 at 37°C for 1 min (0.1 and 1% Triton X-100 groups, respectively). Cryopreserved-thawed whole sperm without any treatment was used as a control. Results from staining with fluorescein diacetate and propidium iodide showed that almost all sperm were propidium iodide positive (dead sperm) immediately after the each treatment. In the control group, approximately 40% of sperm were fluorescein diacetate positive (live sperm) after thawing. The presence of PLCζ (72 kDa) was examined by Western blotting using the antibody against the N-terminal 19-mer sequence of porcine PLCζ (Kurokawa et al. 2005). A band corresponding to porcine PLCζ was not detected in any treatment group in any culture period (from 0 to 135 min). In contrast, PLCζ was detected in the control group and in all culture periods. These results strongly suggest that PLCζ in porcine sperm was lost immediately after the pretreatments, such as by sonication, incubation with 0.1 or 1% Triton X-100, and repeated cycles of freezing–thawing. The decrease in PLCζ protein by pretreatment may be one of the causes of incomplete activation of oocytes in porcine ICSI.

This work was supported by a Grant-in-Aid for JSPS Fellows.
273 SELECTION OF PLURIPOTENT STEM CELLS INDUCED BY XENOPUS EGG EXTRACTS
Authors: Chiang C.-Y. , Tang P.-C.
Published: December 2008

It has been reported that Xenopus egg extracts contain molecules that are capable of reprogramming mammalian somatic cells. The reprogrammed somatic cells, which are called extract treated cells (ETC), possess the potential for clinical therapy as embryonic stem (ES) cells do. Therefore, in addition to establishment of an efficient method to reprogram mouse NIH/3T3 cells by Xenopus egg extracts, the aim of this study was to select the ETC cells by the expression of Oct4. In Experiment 1, two methods, electroporation or permeabilization, were conducted to treat mouse NIH/3T3 cells with Xenopus egg extracts. 2 × 105 cells in 200 μL reprogramming mixture containing Xenopus egg extracts were stimulated by a direct current (DC) pulse (80 V mm–1 for 3 msec) three times followed by a pause of incubation at 37°C for 5 min and a single DC pulse (170 V mm–1, for 0.4 msec) subsequently. The electroporated cells were then incubated at 22°C for 1 h. In the other treatment group, NIH/3T3 cells (5 × 105) were permeabilized by streptolysin O (SLO, 500 ng mL–1 in PBS) for 50 min at 37°C before mixed with Xenopus egg extracts at 22°C for 2 h. Cells were cultured in DMEM supplemented with 10% FBS for the first 4 days and then changed to ES medium (DMEM supplemented with 15% FBS, 0.1 mm β-mercaptoethanol, 1000 unit mL–1 mLIF, 0.5% nonessential amino acids, 2 mm L-glutamine) for the last 6 days after Xenopus egg extract treatment. Cell colonies were found in both treatment groups at the end of culture. Examination by immunocytochemical staining, results showed that the extract-treated cell colonies expressed pluripotent marker proteins, such as alkaline phosphatase, Oct4, Nanog and Sox2. In Experiment 2, an enhanced green fluorescent protein (EGFP) expression vector was constructed and EGFP was driven by Oct4 enhancer and promoter (Oct4-EGFP). Mouse NIH/3T3 cells were then transfected with Oct4-EGFP plasmids and selected for stable clone by G418 screening. After 6 passages, the NIH/3T3-Oct4-EGFP cells were treated with egg extracts to induce reprogramming as Experiment 1, and monitored pluripotency based on the expression of EGFP. Results showed that some of the cells or cell colonies expressed green fluorescence driven by Oct4 regulatory element at the 8th day of culture after extract treatment. Our results demonstrated that both methods of electroporation and reversible permeabilization could introduce reprogramming molecules in Xenopus egg extract to the mammalian somatic cells and generate ETCs cells in vitro. Also, with the establishment of NIH/3T3-Oct4-EGFP cell line, the potentially reprogrammed colonies could be easily selected by EGFP expression. The changes of epigenetic modifications in the ETC cells would be investigated in the short future.
278 EFFECT OF XENOESTROGENS ON THE DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS
Authors: Jeung E.-M. , Choi K.-C. , Jeung E.-B.
Published: December 2008

Endocrine disruptors (ED) may have adverse impacts on reproductive and immune systems in human and wild animals. It has been shown that octyl-phenol (OP) and nonyl-phenol (NP) have estrogenicity in estrogen-responding cells or tissues. In this study, we further investigated the effect(s) of OP and NP on the expression of undifferentiation and differentiation markers in mouse embryonic stem cells (ESC), which function as an important factor in the differentiation of ESC into cardiomyocytes. Mouse ESC were cultured in hanging drops to form embryoid bodies (EB). The medium was replaced with phenol red-free DMEM/F-12 supplemented with 5% charcoal-dextran-stripped FBS. The ESC were treated with OP, NP (1Ã-10-6 and 1Ã-10-7 M) or 17β-estradiol (E2; 1Ã-10-8 and 1Ã-10-9 M) in a time-dependent manner (1, 2 and 3 days), and EB were treated with identical concentrations for 4 and 8 days, respectively. High increasing doses of OP and NP were employed in this study because a binding affinity of ED to estrogen receptors (ER) is about 1000 less than that of E2. We determined the mRNA expression of undifferentiation markers (Oct4, Sox2 and Zfp206) and cardiomyocyte differentiation markers (cardiac alpha-MHC, beta-MHC and myosin light chain isoform-2V) using real-time PCR. In ESC, undifferentiation markers were identified. It is of interest that treatment with OP, NP or E2 induced a significant increase (1.4 5.5-fold) in Oct4 expression at the transcription levels according to a dose- and time-dependent manner. However, no difference was observed in the expression of Sox2 and Zfp206 genes in ESC, suggesting that OP and NP may play a role as an Oct4 enhancer in ESC. In addition, both undifferentiation and cardiomyocyte differentiation markers were identified in EB. Treatment with OP and NP induced a significant increase in the expression of Oct4, Sox2 and Zfp206 genes at the transcription levels in a dose-dependent manner for 4 days, whereas Oct4 expression was only induced at these doses for 8 days. In contrast, cardiomyocyte differentiation markers were reduced by these ED in EB. Taken together, these results suggest that OP and NP play a role as a positive regulator in the undifferentiation process of ESC and EB, and maintenance and differentiation of mouse ESC.
289 COOPERATIVE EXPRESSION OF PLURIPOTENCY-RELATED GENES AND NEURAL CREST MARKER GENES IN PORCINE GFP-TRANSGENIC SKIN-DERIVED PROGENITORS
Authors: Zhao M. T. , Isom C. S. , Zhao J. G. , Hao Y. H. , Ross J. , Whyte J. , Zhang Y. , Prather R. S.
Published: December 2008

Recently neural crest derived multipotent progenitors from skin have attracted much attention as the skin may provide an accessible, autologous source of stem cells available with therapeutic potential (Toma JG et al. 2001 Nat. Cell Biol. 3, 778–784). The multipotent property of stem cells could be tracked back to the expression of specific marker genes that are exclusively expressed in multipotent stem cells rather than any other types of differentiated cells. Here we demonstrate the property of multipotency and neural crest origin of porcine GFP-transgenic skin derived progenitors (termed pSKP) in vitro by marker gene expression analysis. The pSKP cells were isolated from the back skin of GFP transgenic fetuses by serum-free selection culture in the presence of EGF (20 ng mL–1) and bFGF (40 ng mL–1), and developed into spheres in 1–2 weeks (Dyce PW et al. 2004 Biochem. Biophy. Res. Commun. 316, 651–658). Three groups of RT-PCR primers were used on total RNA from purified pSKP cells: pluripotency related genes (Oct4, Sox2, Nanog, Stat3), neural crest marker genes (p75NGFR, Slug, Twist, Pax3, Sox9, Sox10) and lineage specific genes (GFAP, tubulin β-III, leptin). Expression of both pluripotency related genes and neural crest marker genes were detected in undifferentiated pSKP cells. In addition, transcripts for fibronectin, vimentin and nestin (neural stem cell marker) were also present. The percentage of positive cells for Oct4, fibronection and vimentin were 12.3%, 67.9% and 53.7% respectively. Differentiation assays showed the appearance of tubulin β-III positive (39.4%) and GFAP-positive (42.6%) cells in cultures by immunocytochemistry, which share the characteristics of neurons and glial cells, respectively. Thus, we confirm the multiple lineage potentials and neural crest origin of pSKP cells in the level of marker gene expression.

This work was funded by National Institutes of Health National Center for Research Resources RR013438.
29 PRODUCTION OF BOVINE CLONED EMBRYOS BY NUCLEAR TRANSFER USING VITRIFIED IMMATURE OOCYTES AS RECIPIENT CYTOPLASTS
Authors: Forell F. , Feltrin C. , Santos L. C. , Vieira A. D. , Costa U. M. , Hölker M. , Rodrigues J. L.
Published: December 2008

The cryopreservation of immature oocytes is a logistic alternative to make cytoplasts available throughout the year for cloning by somatic cell nuclear transfer (SCNT). Oocyte cryopreservation will help to overcome hurdles related to oocyte availability, seasonality, or sanitary constraints. The objective of this experiment was to determine the efficiency of vitrification of bovine immature oocytes for use as cytoplasts to produce clone embryos. Cumulus–oocyte complexes (COCs) obtained from bovine ovaries by slicing from a local abattoir were selected and vitrified prior to maturation. Vitrification and warming solutions and exposure times were as previously described (Vieira AD et al. 2008 Rep. Dom. Anim. 43, 314–318) with minor modifications. Groups of 15 COCs were loaded in a 5-μL vitrification solution microdrop in beveled-cut straws (0.5 mL), which were plunged into N2L. Following warming, vitrified and control (non-vitrified) oocytes were in vitro-matured for 22 h and 17 h, respectively (Oliveira ATD et al. 2005 Theriogenology 64, 1559–1572). After maturation, cumulus cells were removed and oocytes were selected by the presence of a polar body. Embryo reconstruction by SCNT, carried out by standard micromanipulation procedures using fibroblast cells from adult origin, and in vitro culture to the blastocyst stage (Day 7) were based on our established procedures (Forell F et al. 2008 Acta Sci. Vet. 36, 141–148). Data regarding oocyte recovery following cumulus cell removal, oocyte survival after micromanipulation, and maturation, fusion, cleavage (Day 2), and blastocyst (Day 7) rates were analyzed by the chi-square test. Oocyte recovery (73.0%, n = 558/764 v. 91.4%, n = 529/579), maturation (46.8%, n = 261/558 v. 65.8%, n = 348/529) and cleavage (47.2%, n = 60/127 v. 60.2%, n = 77/128) rates were lower in the vitrified than in the non-vitrified group, respectively (P < 0.05). Conversely, oocyte survival after micromanipulation (77.8% and 78.4%) and fusion (82.1% and 82.3%) and blastocyst (16.7%, 10/60 v. 23.4%, n = 18/77) rates were similar between vitrified and non-vitrified groups. However, the overall efficiency (blastocysts produced from selected COCs) was 3.4-fold lower for vitrified oocytes than controls. In conclusion, the vitrification of immature bovine oocytes was proven as a valuable procedure for the production of blastocysts by SCNT, providing that a strict selection is made following warming, being an alternative resource either for the use of large numbers of oocytes obtained from slaughterhouse ovaries or to overcome seasonal variations in oocyte supply for use in animal cloning.

This work was supported by the Brazilian National Council for Scientific and Technological Development (CNPq).
290 OPTIMAL OF DOSE OF OVINE PITUITARY GLAND EXTRACT OVAGEN® FOR SUPERSTIMULATION OF BEEF COW DONORS IN ARGENTINA
Authors: Brogliatti G. , Villarreal J. , Cutaia L. , Albrech A. , Garcia Guerra A. , Bó G. A.
Published: December 2008

The success of an embryo transfer program is measured by the number of calves born alive by female donor in a given period. The success is influenced by several factors related to the number of ovulations, fertilization rate, and embryo viability (Armstrong D 1993 Theriogenology 39, 7–24). One of the main inconveniences of embryo transfer programs is the variability of superovulatory response to treatments. An experiment was designed to determine the optimal dose of ovine pituitary gland extract Ovagen® (ICPbio, Ltd., Auckland, New Zealand) for inducing superovulation in Aberdeen Angus donor cows. Sixty cycling multiparous donors with a condition score between 3.5 and 4.5 (Scale 1 to 5) were used. All donors received an intravaginal progesterone device DIB (1 g of Progesterone, Syntex, Buenos Aires, Argentina), along with 2 mg of EB (Estradiol Benzoate, Syntex, Buenos Aires, Argentina) and 50 mg of progesterone (Laboratorio Rio de Janeiro, Argentina) on Day 0. Superestimulatory treatment began on Day 4 and donors were randomly assigned to 3 treatment groups according to the total dose of NIADDK-oFSH-Z (Ovagen®) as follows: Group 1, cows received the total dose recommended by the manufacturer (100%* Group), 17.6 mg; Group 2, cows received 75% of the total dose recommended by manufacturer (75% Group), 13.2 mg and Group 3, cows received 50% of the total dose recommended by manufacturer (50% Group), 8.8 mg. All cows received two 150 μg of D+ cloprostenol IM (Ciclase, Syntex) injections on Day 6 given at 12 h interval. DIB was removed on Day 7 a.m. On Day 8 a.m., cows received 0.05 mg of GnRH IM (Gonasyn, Syntex). Fixed time AI was done on Day 8 p.m. and Day 9 a.m. with high quality frozen–thawed semen. On Day 15 embryo collection was performed by non-surgically method and evaluated by developmental stage and quality. The efficiency of superestimulatory response was evaluated by total amount of collected ova-embryos, fertilized ova and embryos Grade 1, 2, and 3, (according to IETS manual). One way AOV test was used to compare variables among groups and results are shown in Table 1. A significant increase in total CL was observed for the groups receiving 75% or 100% of the recommended dose with respect to the group receiving 50% of that dose. There was also a significant greater number of grade 1 embryos for the groups receiving 75% or 100% of the dose than in the group receiving 50% of the recommended dose. There were no differences for any of the evaluated parameters between 75% and 100% dose groups. These results suggest that acceptable superstimulatory responses can be obtained using reduced doses of Ovagen® in Aberdeen Angus donor cows. (*) the percentage makes reference to the total dose recommended by the laboratory of origin.

Table 1.Embryo collection results of superstimulated donors with different dose* of NIADDK-oFSH-Z (Ovagen)

Research supported by Syntex S.A. and Eolia S.A.
302 CONFIRMATION OF GERM-LINE TRANSMISSION OF RED FLUORESCENCE PROTEIN TRANSGENIC MALE CAT
Authors: Cho S. J. , Lee Y. S. , Choi E. G. , Bang J. I. , Yu X. F. , Kong I. K.
Published: December 2008

Production of transgenic animals is highly desirable for biotechnology and basic research. Therefore, a method of producing genetically modified cats through genetic engineering is important for generating biomedical models of human diseases. We investigate reproductive ability of red fluorescence protein (RFP) transgenic cloned male cat (RFP TG cat) in natural mating with domestic female cat. One domestic female cat in natural mating with RFP TG cat delivered 6 kittens of which 3 (2 female and 1 male) showed RFP expression of parental line. Among the 3 RFP expressing kittens, 1 died at day 5 and were subjected to PCR analysis for confirmation of RFP gene expression. Red fluorescence protein gene was detected in tissue samples including liver, muscle, brain, large intestine, bladder, spleen, sexual organ, small intestine, kidney, heart, stomach, pancreas, lungs, and skin of the kitten. These results indicated that RFP transgenic cloned male cat have normal reproductive fertility, and the stability of transmission of the RFP transgene through the germ line and its normal expression in offspring.

Supported by KOSEF (M10525010001-05N2501-00110). S. J. Cho, Y. S. Lee, E. G. Choi, and J. I. Bang were supported by a scholarship from the Post BK21 Program, the Ministry of Education, Science and Technology, Korea.
306 GENERATION AND CHARACTERIZATION OF HUMAN A20 TRANSGENIC PIGS BY SOMATIC CELL NUCLEAR TRANSFER
Authors: Oropeza M. , Petersen B. , Lemme E. , Lucas-Hahn A. , Queisser A. L. , Herrmann D. , Hassel P. , Niemann H.
Published: December 2008

Xenotransplantation is considered a solution to diminish the acute shortage of human organs. Although the hyperacute rejection occurring instantly after xenotransplantation can already be reliably controlled, the following immunological defense like the acute vascular rejection (AVR) remains a major hurdle for long-term survival of xenografts in porcine-to-primate organ transplantation. AVR is primarily characterized by endothelial cell activation with severe consequences on coagulation. The human A20 (hA20) gene exhibits antiapoptotic and anti-inflammatory properties in endothelial cells (Ferran C 2006 Transplantation 82, 36–40) and could thus prevent endothelial cell activation leading to AVR and xenograft destruction. Here, hA20-transgenic pigs were produced by somatic cell nuclear transfer (SCNT) in order to examine the ability of hA20-expressing tissues and organs to modulate the AVR. Two hA20-expression vectors, containing the promoters CAGGS or EF1-α in addition to a neomycin resistance cassette, were transfected into porcine fetal fibroblasts. Transfection was accomplished by electroporation, and the cell clones were selected with G418 (800 μg mL–1) for 14 days. Resistant cell clones were screened in PCR with hA20-specific primers. SCNT was performed as previously described (Hölker M et al. 2005 Cloning Stem Cells 7, 35–44). After 8 SCNT sessions with pCAGGSEhA20-transgenic cell clones, embryo transfer was carried out to 16 peripuberal recipients resulting in 12 pregnancies (75%). Sixteen fetuses were isolated after sacrificing the recipient sows, and 45 piglets were born. Six of 16 fetuses (37.5%) and 15 of 45 (33.3%) piglets were transgenic. Four SCNT sessions were completed with pEF1hA20-transgenic cell clones following embryo transfer to 6 sows and 5 pregnancies were established (83.3%). Five fetuses were isolated and 14 piglets born. Five of 5 fetuses (100%) and 9 of 14 (64.3%) piglets were transgenic. Expression analysis (RT-PCR and Northern blots) showed transcription of the hA20 gene in heart, muscle, and cultivated porcine aortic endothelial cells of the pCAGGSEhA20-transgenic animals. No transcription was detected in pEF1hA20-transgenic animals. Current results show that hA20-transgenic pigs develop physiologically compared to wildtype counterparts. Expressing tissues and organs were only found in pCAGGSEhA20-transgenic animals, which are now being tested regarding functionality of the hA20 transgene.

We would like to thank Prof. Beyaert from the University of Ghent, Belgium, for providing us with the A20-expression vector pCAGGSEhA20.
35 DEVELOPMENT OF INTERSPECIES CLONED MOUSE EMBRYOS RECONSTRUCTED WITH PORCINE ENUCLEATED OOCYTES
Authors: Jiang Y. , St John J.
Published: December 2008

Interspecies somatic cell nuclear transfer (iSCNT) offers significant opportunities for the derivation of embryonic stem cells (ESCs) that can model diseases. In this study, we investigated the development of murine-porcine iSCNT embryos and compared these to outcomes associated with porcine-porcine SCNT embryos. Enucleation was performed on in vitro-matured porcine oocytes using the Oosight spindle viewer system (CRI, UK). Either murine embryonic fibroblasts or porcine cumulus cells were injected into porcine ooplasm using a piezo-electric micromanipulator. After being exposed to the porcine ooplasm for 1 h, the reconstructed oocytes were activated by a combination of A23187 and 6-DMAP for 3.5 h. For the murine-porcine embryos (n = 9375), we used a serial culture method, namely porcine zygote medium (PZM) for 48 h followed by incubation with KSOM for another 5 days. We found that the majority of murine-porcine embryos (73.58%) arrested at the 4- to 8-cell stage, whilst a very small minority progressed through to blastocyst (0.064%). Porcine-porcine SCNT reconstructed oocytes (n = 142) cultured in PZM achieved a 15% blastocyst development rate. In the murine-porcine embryos, 10/44 embryos had chromosomal DNA loss associated with apoptosis, as determined by immunocytochemistry using the H2A.X antibody. We also found that at later stages, morula to blastocyst, the mouse-to-pig reconstructed embryos did not exhibit murine-specific Oct-4 expression patterns. Furthermore, murine mitochondrial DNA tended to be eliminated rather than preserved by the murine nuclei, which could render any subsequently derived ESCs dysfunctional due to 2 diverse genomes contributing genes to the electron transfer chain.
39 FACTORS AFFECTING PREGNANCY RATE FOLLOWING TRANSFER OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS IN DOGS
Authors: Kim M. J. , Oh H. J. , Park J. E. , Kang J. T. , Hong S. G. , Goo O. J. , Kim D. Y. , Lee B. C.
Published: December 2008

There have been attempts to find out factors influencing pregnancy rate after transfer of somatic cell nuclear transferred (SCNT) couplets in several species. The purpose of this study was to investigate the effects of parity, surgical history, and synchronization state between oocytes donor and recipient on the pregnancy rate following transfer of SCNT embryos in dogs. A total of 25 mixed-breed female dogs, aged 1 to 5 years old, weighing 20 to 35 kg were used in this study. In vivo-matured oocytes, obtained by flushing uterine tubes of donor dogs approximately 72 h after ovulation, were used for SCNT. Ovulation was determined by daily serum progesterone concentration. Reconstructed embryos (range = 7–36, average = 18.0 embryos) with normal morphology were surgically transferred into the ampullary portion of the uterine tube of recipient dogs. Pregnancy was determined at least 26 days after transfer by untrasonography. In experiment 1, recipient females were divided into 2 groups: nulliparous (7 bitches) and multiparous (17 bitches) groups. In experiment 2, recipient females were divided into 2 groups by surgical history: operated (10 bitches) and non-operated (14 bitches) groups. Operated group included those with a history of 1 laparotomy, either flushing of uterine tubes or SCNT embryo transfer. Non-operated group included those without history of any surgery. In experiment 3, recipient females were divided into 2 groups: synchronous (13 bitches) and asynchronous (7 bitches) groups. Synchronous was defined that the oocyte donor dog and the recipient dog ovulate at the same day. Asynchronous was defined that the oocyte donor dog ovulates 1 day earlier than the recipient. Data were analyzed using the Statistical Analysis System program (SAS Institute, version 9.1, Cary, NC, USA). In experiment 1, pregnancy rate was significantly higher in the multiparous group than the nulliparous group (23.5 v. 14.2%, P < 0.05). In experiment 2, surgical history did not influence the pregnancy rate between operated and non-operated groups (20.0 and 21.4%, respectively). In experiment 3, pregnancy rate was significantly higher in the asynchronous group than the synchronous group (42.9 v. 15.4%, P < 0.05). In conclusion, multiparous bitches improved the efficiency of SCNT embryo transfer. A higher pregnancy rate resulted when the oocyte donor dog’s ovulation time was 1 day earlier than that of the recipient dog. Other factors should be investigated for further improvement of the SCNT embryo transfer efficiency.

This study was financially supported by the Korean MEST, through KOSEF (grant # M10625030005-08N250300510) and the BK21 program for Veterinary Science, and SNU foundation (Benefactor; RNL BIO).
4 THE HISTONE METHYLTRANSFERASE Suv39h2 ADOPTS A NUCLEAR LOCALIZATION DURING CLEAVAGE DEVELOPMENT IN PARTHENOGENETIC PORCINE EMBRYOS
Authors: Johnson C. M. , Cabot R. A.
Published: December 2008

Successful cleavage development of mammalian embryos requires precise activation and repression of transcription. Covalent modifications to histone proteins, such as methylation and acetylation, play a key role in transcriptional regulation. In particular, dimethylation of the lysine 9 residue of histone protein H3 (H3K9) results in gene silencing and heterochromatin formation. Our lab has previously shown that transcripts encoding the five histone methyltransferases known to methylate H3K9 (Suv39h1, Suv39h2, ESET, G9a, and EHMT1) are present in different amounts during oocyte maturation and cleavage development. Specifically, Suv39h2 is in the greatest abundance in GV and metaphase II stage oocytes and is also present throughout cleavage development. The aim of this study was to determine the localization of Suv39h2 protein in the GV-stage oocyte and pronuclear, 2 cell, and 4 cell stage parthenogenetic porcine embryos. We hypothesized that Suv39h2 protein would localize to the nucleus based on its high transcript abundance throughout cleavage development. To test this hypothesis, we performed a microinjection experiment in which mRNA encoding a porcine Suv39h2-GFP fusion protein was injected into metaphase II porcine oocytes. Porcine oocytes were matured in a defined medium (TCM-199 supplemented with 0.1% PVA, 0.069 mg mL–1 cysteine, 10 ng mL–1 EGF, 0.5 IU mL–1 LH and FSH) for 42 to 44 h at 39°C in 5% CO2, then denuded of cumulus cells just before microinjection. Two separate treatment groups were microinjected intracytoplasmically with 1 μg μL–1 GFP or Suv39h2-GFP mRNA, respectively. Microinjection was performed using a FemtoJet microinjector (Eppendorf, Hamburg, Germany). The treatment groups and non-injected controls were electroactivated independently and cultured in PZM medium supplemented with 3 mg mL–1 BSA for 12 (pronuclear), 24 (2 cell), or 48 (4 cell) hours at 39°C in 5% CO2, depending on desired stage of development. Before visualization under UV light, embryos were stained with Hoechst 33342 for 15 minutes. Oocytes and embryos were analyzed for GFP expression at the GV, pronuclear, 2 and 4 cell stages of development using epifluorescence microscopy. Two to four biological replicates were performed for each stage of embryo development. We found that Suv39h2-GFP protein showed nuclear localization in most GV-stage oocytes (n = 11/14) and pronuclear (n = 17/17), 2-cell (n = 34/36), and 4-cell (n = 9/9) stage embryos. Chi square analysis revealed this pattern to be different from that observed in embryos injected with GFP mRNA, where GFP did not display nuclear localization at any stage of development (n = 12; P < 0.05). These results indicate that Suv39h2 is localized in the nucleus of oocytes and cleaved embryos, which suggests that this histone methyltransferase plays an important role in methylating H3K9.
41 PRODUCTION OF CLONED MINIATURE PIGS USING BONE MARROW MESENCHYMAL STEM CELLS
Authors: Lee S. L. , Kang E. J. , Jeon B. G. , Park J. K. , Hyun S. H. , Joo Y. K. , Sung H. W. , Lee E. S. , Rho G. J.
Published: December 2008

The cloning of miniature pigs (Mini-pig) are considered to advance in genetic engineering technology and xenotransplantation. A few researches have recently been reported successfully produce cloned Mini-pigs using somatic cells, however its efficiency is still low. The present study was aimed to successfully produce cloned Mini-pigs derived from bone marrow mesenchymal stem cells (MSCs) by NT, and compared the developmental ability of cloned Mini-pigs between fetal fibroblast (FF) and differentiated MSCs. For the production of the cloned Mini-pig derived from MSCs, MSCs were isolated from a 1 month old of female Mini-pigs (T-type, PWG Micro-pig®, PWG Genetics Korea, Ltd.). MSCs were differentiated into osteocytes, adipocytes, and chondrocytes under controlled conditions and characterized by cell surface antigen profile using specific markers. These differentiated or undifferentiated MSCs, FFs of Mini-pig were transferred into enucleated oocytes of domestic pigs, and 2-cell stage of 100 NT embryos were surgical transferred to the synchronized recipients. Statistical analysis was performed using one-way ANOVA, t-test, Duncan’s and Tukey’s multiple comparisons test by SPSS (SPSS, Inc., Chicago, IL, USA). The developmental potential of NT embryos derived from MSCs (differentiated and undifferentiated), the rates of blastocyst formation was significantly (P < 0.05) higher than NT embryos derived from FFs, however the NT embryos derived from three different types of differentiated MSCs were significantly (P < 0.05) lower than undifferentiated MSCs. In addition, total cell numbers in NT blastocysts derived from MSCs were significantly (P < 0.05) higher than NT blastocysts derived from FFs, but it did not significantly (P < 0.05) differ between differentiated or undifferentiated MSCs. NT embryos derived from MSCs were transferred to 5 domestic pig recipients, and 5 cloned Mini-pigs were obtained from 2 recipients (one stillbirth and 4 viable offspring). All of them were confirmed by the microsatellite analysis (8 markers) of the genomes of cloned offspring, donor MSCs and recipients. Physical and histological studies are in the process for the characterization of a cloned Mini-pig derived from MSCs as animal model. The results demonstrated that, in vitro developmental ability of NT embryos derived from undifferentiated MSCs were a higher than those from differentiated MSCs or FFs. Moreover, multipotent MSC might have a potential for the production of viable cloned Mini-pigs. Therefore, MSCs as a nuclear donor might a key to improving the production of cloned Mini-pig as animal model for xenotransplantation.

This work was supported by Grant No. 20070301034040 from Bio-organ, Republic of Korea.
46 EFFECT OF SHORT-TERM TRICHOSTATIN A TREATMENT ON BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS
Authors: Oh H. J. , Park J. E. , Kim M. J. , Hong S. G. , Kang J. T. , Kim D. Y. , Jang G. , Lee B. C.
Published: December 2008

Epigenetic reprogramming such as acetylation in somatic cell nuclear transfer (SCNT) has been known as one of problems in cloned embryos. For resolving this acetylation reprogramming, many investigators recently have reported the effect of long-term culture of post-activated SCNT embryos using trichostatin A (TSA), a histone deacetylase inhibitor (HDACi). The objective of this study is to investigate the effect of short-term TSA treatment on in vitro developmental ability and the quality of bovine SCNT embryos. Immature oocytes were aspirated from abattoir-derived ovaries, matured in vitro for 22 h, and enucleated. A bovine fetal fibroblast was placed into the enucleated oocyte and fused by electrical stimulation. The fused couplets were activated by 4-min incubation in 10 μm ionomycin, followed by 4 h of culture in 1.9 mm 6-dimethylaminopurine with or without TSA (0, 50, or 100 nm). The SCNT embryos were subsequently cultured in modified synthetic oviduct fluid medium for 8 days. Developmental competence was assessed by blastocyst formation and total cell number. Total cell numbers were determined by staining with bisbenzimide 33342. As results, developmental competence to blastocysts was higher in 100 nm than control (36.7 v. 27.9%, P < 0.05). In blastocyst hatching rate, TSA 100 nm group (19.5%) at 8 days showed an increased pattern as opposed to control and TSA 50 nm group (11.1 and 12.7%; P < 0.05). No significant differences in two cell and morula stage were observed among treatment groups. In terms of development to hatching stage of blastocysts, TSA 100 nm group (19.5%) at 8 days has a significant effect compared to control and TSA 50 nm group (11.1 and 12.7%; P < 0.05). Total cell number of blastocysts derived from TSA 100 nm was significantly higher (P < 0.05) than that in TSA 50 nm (116 v. 100), whereas there was not significant difference between control and TSA 100 nm. In conclusion, short-term culture with high concentration of TSA improved the blastocysts formation however total cell number of blastocysts showed contradictory result. The epigenetic modification by TSA treatment on bovine SCNT needs further investigation.

This study was financially supported by KOSEF (grant # M10625030005-08N250300510) and the Korean MEST, through the BK21 program for Veterinary Science.
56 EFFECTS OF KNOCKDOWN OF DNA METHYLTRANSFERASE 1 BY RNA INTERFERENCE ON IN VITRO DEVELOPMENT AND DNA METHYLATION STATE IN BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS
Authors: Yamanaka K. , Sakatani M. , Takahashi M.
Published: December 2008

Reprogramming of epigenetic modification is a necessary process during mammalian development, which is aberrant in somatic cell nuclear transfer (SCNT) embryos. Previous study has demonstrated that an abnormal state of genomic hypermethylation is consistently observed in SCNT embryos (Kang et al. 2001 Nat. Genet. 28, 173–177). On the other hand, small interfering RNAs (siRNAs) are identified molecules shown to silence genes via targeted mRNA degradation and are widely used in molecular and cellar research (Hannon GJ 2002 Nature 418, 244–251). Thus, knockdown of the expression of genes related to epigenetic modifications by siRNA may be used to alter epigenetic modifications in SCNT embryos. In the present study, we investigated the effects of knockdown of DNA methyltransferase 1 by siRNA on in vitro development, gene expression, and DNA methylation state of bovine SCNT embryos. In vitro matured oocytes were enucleated, fused with bovine fibroblasts and then activated, the resultant SCNT embryos were divided into three groups; control, non-treated group; sham-NT, H2O injected group; and siRNA-NT, siRNA injected group. The siRNA corresponding to DNA methyltransferases 1, which is the enzyme responsible for maintaining DNA methylation patterns, was designed and injected into the cytoplasm of SCNT embryos. All embryos were cultured in CR1aa + 5% FCS and assessed the rates of cleavage and blastocyst formation on Days 2 and 8, respectively. All data were obtained from more than 5 replicates. Developmental percentage data were analyzed by chi-square tests (P < 0.05). Other data were analyzed with ANOVA followed by Fisher’s protected least significant difference (P < 0.05). The developmental rate to blastcysts in siRNA-NT group (38.7%; 111/287) was significantly higher (P < 0.05) than those of control (28.8%; 121/420) and sham groups (30.5%; 92/302). To estimate the effect of siRNA injection on gene expression, we sampled embryos at 48 h after culture and measured the amount of DNA methyltransferase 1 mRNA expression by real-time PCR. The amount of DNA methyltransferase 1 mRNA was significantly less (P < 0.05) than those of control and sham-NT groups. Finally, the levels of DNA methylation at satellite I region were analyzed by COBRA method in blastosyst stage embryos. The level of DNA methylation of blastocysts in siRNA-NT groups was significantly less (P < 0.05) than those of control and sham-NT and also similar to that of IVF blastocysts. In the present study, we showed that gene silencing of DNA methyltransferase 1 by siRNA enhanced the in vitro development of SCNT embryos and decreased the level of DNA methylation which was equivalent to IVF embryos. These findings suggest that knockdown of specific genes related epigenetic modifications by RNA interference may alter abnormal epigenetic reprogramming with the resultant improvement for subsequent development of SCNT embryos.
61 PLANT EXTRACTS REDUCE DNA FRAGMENTATION IN FROZEN-THAWED STALLION SPERM
Authors: Burns P. D. , Wong N. , Arnold H. , Sirs N. , Romero R. , Herickhoff L.
Published: December 2008

Mares inseminated with frozen–thawed sperm have reduced pregnancy rates compared with mares inseminated with fresh sperm. Processing mammalian sperm for cryopreservation increases the concentration of free radicals and induces oxidative stress, which can result in DNA damage and may lead to lower fertility. The objective of this experiment was to examine the effects of several plant antioxidant extracts on stallion sperm post-thaw motility and DNA quality. Single ejaculates were collected from 4 stallions and the concentration of sperm cells in each ejaculate was determined spectrophotometrically. Semen was centrifuged at 300g for 10 min at room temperature and seminal plasma was removed. Sperm pellets were resuspended to a final concentration of 200 × 106 cells mL–1 in E-Z Freezin LE (ARS, Chino, CA) extender (control) or extender containing 1 of 3 plant extracts (3% v/v) from 2 different commercial sources. Extended sperm cells were loaded into 0.5-mL straws and frozen over liquid nitrogen vapor for 10 min. Straws were then plunged into liquid nitrogen and stored until further evaluation. Motility and velocity parameters were determined at 0, 30, and 60 min post-thaw using a computer-assisted sperm analyzer. DNA fragmentation was determined immediately after thawing using a single-cell gel electrophoresis (Comet) assay. Motility (total and progressive) and velocity parameters of sperm cells did not differ between controls and plant extract treatments (P > 0.05). However, total Comet length and tail length were reduced in sperm cells stored in extender containing each plant extract (P < 0.05). Tail and olive moment tended to be reduced (P < 0.10) in sperm cells stored in plant extracts. In conclusion, sperm cells stored in plant extracts had reduced post-thaw DNA damage. The addition of plant extracts to commercial freezing extenders may be a practical method for improving sperm quality.
69 A HOMEMADE N2 SUPERCOOLING DEVICE (NITROCOOLER) ENHANCES VIABILITY AFTER VITRIFICATION OF BOVINE OOCYTES BUT NOT OF IN VITRO-PRODUCED EMBRYOS
Authors: Mezzalira J. C. , Vieira A. D. , Zanardi G. , Gonçalves M. C. , Rodrigues M. F. , Martins L. T. , Ohlweiler L. U. , Bertolini M. , Mezzalira A.
Published: December 2008

Increasing the cooling rate is a common strategy to enhance vitrification efficiency of bovine oocytes and in vitro-produced (IVP) embryos. Under vacuum conditions, liquid N2 (LN2) temperature decreases, increasing the cooling rate during the vitrification procedure. However, commercially available brands of equipment to supercool nitrogen are expensive. The aim of this study was to verify the effectiveness of a low-cost homemade nitrogen supercooling apparatus (Nitrocooler) for vitrification of bovine oocytes and IVP embryos. The device consists of a vacuum pump coupled to a close-tight-lidded flask with a styrofoam cup filled with 300 mL of LN2. After 5 to 7 min of vacuum pumping, LN2 goes through the slushing phenomenon, turning solid. After the lid is opened, the N2 turns liquid again, but in a stable, supercooled physical state lasting for approximately 10 min, boiling off when objects are plunged into it. Nitrocooler was tested for vitrified oocytes (Experiment I), vitrified oocytes in different containers (Experiment II), and embryos in 2 different vitrification solutions (Experiment III). In Experiment I (Ciência Rural, 2006 36, 1501–1506), immature (IM, n = 172) or in vitro-matured (IVM, n = 174) oocytes were exposed to 10% ethylene glycol (EG) + 10% DMSO for 30 s, followed by 20% EG + 20% DMSO + 0.5 m sucrose for 20 s, loaded in open-pulled straws (OPS), and plunged into LN2 or Nitrocooler-pumped LN2. For both IM (8.8%) and IVM (10.6%) oocytes, Nitrocooler-pumped LN2 increased blastocyst rates (Bonferroni, P < 0.05). In Experiment II, 1454 oocytes were vitrified using Nitrocooler after loading in different containers: stainless steel straws (SSS), beveled-tip straws (BTS), open-OPS, and glass-pulled straws (GPS). Greater blastocyst rates were obtained in the SSS (10.2%) and the GPS (8.0%) treatments, both being greater than the BTS (6.1%) and OPS (6.1%) containers (Tukey, P < 0.05). In Experiment III (Acta Sci. Vet. 2006 34, 77–82), 195 IVP bovine blastocysts were vitrified with or without Nitrocooler under 2 different cryoprotectant solutions: 10% EG + 10% DMSO or 10% EG + 10% propylene glycol (PROP). Hatching rates using Nitrocooler for EG + DMSO (50.2%) or EG + PROP (54.0%) were similar (ANOVA, P > 0.05) to normal atmosphere rates using EG + DMSO (50.1%) or EG + PROP (56.0%). Under these experimental conditions, our results suggest that the greater cooling rates obtained favored an increase in oocyte viability after vitrification, whereas no such beneficial effects were detected for vitrified IVP embryos. In conclusion, Nitrocooler was proven effective as a low-cost device to supercool LN2, increasing viability after vitrification of bovine oocytes, but not of IVP embryos.

Table 1.Effect of Nitrocooler apparatus on cryopreservation of bovine oocytes and embryos