bluuebunny/crossref_metadata_2025 huggingface dataset:
190 EFFICIENT GENERATION OF INDUCED PLURIPOTENT STEM CELLS FROM PORCINE ADIPOSE-DERIVED STEM CELLS WITH A FEEDER-INDEPENDENT AND SERUM-FREE SYSTEM
Authors: Zhang Y. , Wei C. , Zhang P.-F. , Li X. , Li Y.-S. , Zhang Y.-L. , Zhang Y.-H.
Published: December 2013

Somatic cells could be directly reprogrammed into stem state by ectopic expression of transcription factors, which share similar features of embryonic stem cells (ESC). Induced pluripotent stem cells (iPSC) possess promising application in producing genetically modified animals, whereas the generation of porcine offspring from iPSC is still difficult and controversial, and new materials are needed. In this study, we report the generation of iPSC from porcine adipose-derived stem cells (pADSC) using drug-inducible expression of defined human factors (Oct4, Sox2, Klf4, and c-Myc) and ‘2i’ plus leukemia inhibitory factor (LIF) culture system. pADSC were isolated from subcutaneous adipose tissue of a 28-day-old Danish Landrace, and subsequently characterised by high proliferation rate at low passages, long period passaging without significant replication senescence, mesenchymal stem cell-specific surface markers expression, including CD29 (0.995 ± 0.0577), CD44 (0.999 ± 0.0333), and CD90 (0.994 ± 0.0333), together with successful adipogenic and osteogenic differentiation ability in vitro. The reprogramming of iPSC from pADSC was evidently more efficient than the process from adult fibroblasts (P < 0.01), both of which were carried out under feeder-independent and serum-free conditions, and this may be due to the higher demethylation level of genomic DNA in pADSC. Two lines of porcine iPSC with naïve-like state were finally obtained through feeder-independent and serum-free conditions. The successful reprogramming of iPSC was demonstrated by short cell cycle interval, alkaline phosphatase (AP) staining positive, expression of stemness-related proteins including OCT-4, SOX2, NANOG, SSEA3, and SSEA4. Full reprogramming of iPSC was evaluated by the significant up-regulation of LIN28, ESRRB, UTF1, and DPPA5. Naïve-like state of porcine iPSC was further confirmed by the striking resemblance to naïve mESC, single-cell dissociation, LIF-dependency, up-regulation of STELLA and ERAS, and little translation of TRA-1-60 and TRA-1-81. In addition, porcine naïve-like iPSC possessed normal karyotypes, and could differentiate into cell types of all three germ layers in vitro and in vivo. Furthermore, in vivo studies to determine the capacity of these cells to integrate into the inner cell mass of blastocysts are still being undertaken for validation. Together, our study provided an efficient method to derive porcine naïve-like iPSC from pADSC, which may be useful for the production of living offspring.
Y. Zhang and C. Wei contributed equally to this work. Y.-H. Zhang is the corresponding author. This work was supported by the National Natural Science Foundation Program 31272442.
193 HYALURONIC ACID-GLYCIDYL METHACRYLATE HYDROGELS SUPPORT IN VITRO CHONDROGENIC DIFFERENTIATION OF PORCINE ADIPOSE-DERIVED STEM CELLS
Authors: Rabel R. A. C. , Osterbur L. , Maki A. , Lewis J. , Wheeler M. B. W.
Published: December 2013

There is a great need for bioengineered cartilage because of the lack of medical or surgical therapies to improve articular cartilage healing. We hypothesised that porcine adipose-derived stem cells (pASC) can be induced to undergo chondrogenic differentiation within hyaluronic acid (HA) hydrogels. The objective of this study was to develop UV-curable pASC-laden HA hydrogels aimed at application in cartilage tissue engineering. HA was treated with glycidyl methacrylate (GM) to allow chemical gelation of the polymer upon exposure to UV light. 2% HAGM hydrogel was obtained by mixing HAGM with chondrogenic medium consisting of TGFβ, ascorbic acid, ITS+ premix (insulin, transferrin, selenous acid; Cat. No. 354352, BD Biosciences, Franklin Lakes, NJ), sodium pyruvate, and dexamethasone. Passage three-pASC were resuspended in 2% HAGM hydrogel with 2 × 107 cells mL–1. Twelve-and-one-half (12.5)-μL droplets (micromasses) of this suspension containing 250 000 pASC were placed in 24-well culture plates and incubated for 2 h at 37°C and 5% CO2 to allow for cell attachment. Subsequently, the cell-laden hydrogels were cured with ~10 mW cm–2 365-nm UV light for 10 min, covered with 500 μL of chondrogenic medium, and cultured for up to 11 days at 37°C and 5% CO2. Additionally, pASC micromasses were cultured in chondrogenic medium without loading on 2% HAGM hydrogels as positive controls, and in non-chondrogenic DMEM as negative controls. Samples were collected at 4, 7, and 11 days in to culture for cryopreservation (for immunohistochemistry; IHC) and dimethylmethylene blue (DMMB) assay. IHC on day 11 of culture demonstrated the expression of cartilage specific proteins type-II collagen and aggrecan. On the basis of data from the DMMB assay, chondrogenic differentiation of pASC-laden micromasses in positive controls and 2% HAGM treatments were not different (P > 0.05). This indicates that ASC can produce cartilage equally well under both conditions, supporting the idea that HAGM may be used as a matrix for cartilage formation in vitro and possibly in vivo. In conclusion, using a micromass cell culture system, we demonstrated that 2% HAGM hydrogels support proliferation and chondrogenic differentiation of pASC. Further experiments testing different concentrations of HAGM and UV exposure levels, and larger sample numbers are warranted to further improve this procedure.
195 EFFECTS OF GSK3 INHIBITOR ON THE PLURIPOTENCY MAINTENANCE OF BUFFALO EMBRYONIC STEM-CELL-LIKE CELLS
Authors: Lu F. , Lao Y. , Sun H. , Lei C. , Deng Y. , Luo C. , Jiang J. R. , Shi D. S.
Published: December 2013

In this study, to explore the effects and mechanism of Wnt/β-catenin signalling pathway on the maintenance of pluripotency of buffalo embryonic stem-cell-like cells (buffalo ESC-like cells), the GSK3 inhibitors BIO and CHIR99021 were added throughout the experiment – i.e. from buffalo inner cell mass (ICM) culture to ESC-like line generation. The buffalo ICM were respectively cultured in the medium containing 0.5 μg mL–1 BIO and 5 mmol L–1 CHIR99021. The percentage of ICMs attachment and primary colony formation were observed, and found that there was no significant difference in the ICMs attachment rate among of the BIO, CHIR99021, and the control groups (91.18% and 92.98% v. 94.59%; P > 0.05). Treating ICMs with CHIR99021 resulted in more primary colony formation rate compared with the control group (77.71% v. 55.41%; P < 0.05). The proliferation rate of primary colonies of buffalo ESC-like cells was detected by bromodeoxyuridine immunofluorescence techniques. The results show that the proliferation rate of primary colonies in the group of buffalo ESC-like cells treated with CHIR99021 was significantly higher than that of the control group on Day 1, Day 3, Day 4, and Day 5 (P < 0.05), and it was also evidently higher than that of control group only on Day 1 (P < 0.05) in the group of BIO, but there was no significant difference in other days (P > 0.05). The mRNA expression level of proliferation marker PCNA of ESC-like cells was significantly up-regulated in both CHIR99021 and BIO treatment groups (P < 0.05), however, treating buffalo ESC-like cells with CHIR99021 significantly up-regulated the expression of pluripotent gene Oct4 and Sox2 (P < 0.05), but had no effect on pluripotent gene Nanog expression (P > 0.05). Oct4 expression was significantly increased (P < 0.05), and the expression of Sox2 and Nanog were significantly decreased (P < 0.05) in the group of BIO treatment. Furthermore, the relative protein level of β-catenin (the downstream effector of Wnt/β-catenin signalling pathway) and the mRNA expression level of c-Myc (the downstream target gene of Wnt/β-catenin signalling pathway) were significantly increased when buffalo ESC-like cells respectively treated with CHIR99021 and BIO (P < 0.05). In conclusion, treating buffalo ESC-like cells with GSK3 inhibitors CHIR99021 can promote proliferation of buffalo ESC-like cells, maintain their undifferentiated state, and up-regulate the expression levels of β-Catenin and c-Myc in buffalo ESC-like cells. These results indicate that Wnt/β-catenin signalling pathway plays an important role in regulation of self-renewal of buffalo ESC-like cells.
This work was funded by the China High Technology Development Program (2011AA100607), China Natural Science Foundation (31072033), and Guangxi Science Foundation (2012GXNSFFA060004).
196 EPITHELIAL–MESENCHYMAL TRANSITION IN EQUINE AMNIOTIC PROGENITOR CELLS INDUCES CHANGES OF THE CELL GLYCAN PROFILE
Authors: Lange-Consiglio A. , Accogli G. , Cremonesi F. , Desantis S.
Published: December 2013

Epithelial to mesenchymal transition (EMT) is the process by which epithelial cells dramatically alter their shape and motile behaviour as they differentiate into mesenchymal cells. The EMT and the reverse process, termed mesenchymal–epithelial transition, play central roles in embryogenesis. Gastrulation and neural crest formation are processes governed by EMT in amniotes. It is noteworthy that in placental mammals, the epithelial layer of amnion originates from the trophectoderm and it is continuous with the epiblast. On this basis, it is reasonable to speculate that some amniotic epithelial cells may escape the specification that accompanies gastrulation, and may retain some of the characteristics of epiblastic cells, such as pluripotency, behaving as stem cells that are able to preserve intrinsically the ability to transdifferentiate. Because it seems that malignant cells use the same mechanisms during the formation of tumours in vivo, the amniotic epithelial cells (AEC) could represent a good model to study in vitro this phenomenon that we observed to occur spontaneously in our culture conditions. The aim of this study was to characterise the glycoprotein pattern expressed in fresh or cryopreserved equine AEC, mesenchymal (AMC), and transdifferentiated cells by means of lectin histochemistry. AEC and AMC were cultured until passage (P) 3, while transdifferentiated cells at P1(EMT1) and P2 (EMT2). All cell lines were frozen for 1 month at –196°C in liquid nitrogen. The glycoanalysis was performed with a panel of twelve lectins to detect the glycans terminating with sialic acids (MAL II, SNA, PNA after sialidase digestion (K-s), K-s-DBA), galactose (PNA, RCA120, GSA I-B4,), N-acetylgalactosamine (DBA, HPA, SBA), N-acetylglucosamine (GSA II), fucose (UEA I, LTA), or with internal mannose (Con A). After freezing: 1) AEC exhibited decrease of binding sites for DBA, SBA, HPA, GSA II, and disappearance of GSA I-B4 and UEA I binders; 2) AMC displayed increase of SBA reactivity, decrease of K-s-PNA, HPA, GSA II staining, and absence of GSA I-B4 affinity; 3) EMT1 cells showed the appearance of K-s-DBA staining, the increase of K-s-PNA, RCA120, SBA, GSA I-B4, and UEA I reactivity, the decrease of MAL II, SNA, HPA, GSA II binders, and the disappearance of DBA and LTA binding sites; 4) EMT2 cells revealed the increase of K-s-PNA, GSA I-B4, UEA I affinity, the decrease of MAL II, SNA, RCA120, HPA, GSA II binders, and the lack of DBA, SBA, and LTA reactivity. In conclusion, this study demonstrates that the EMT induces changes in cell surface glycan profile of equine amniotic progenitor cells, and for the first time revealed that freezing modifies the lectin binding pattern of these cells. The observed glycan pattern modification may represent one aspect of the spontaneous complex process of EMT.
199 GERM-CELL-LIKE CELLS GENERATION FROM GOAT INDUCED PLURIPOTENT STEM CELLS
Authors: Singhal D. K. , Malik H. N. , Singhal R. , Saugandhika S. , Dubey A. , Boateng S. , Kumar S. , Kaushik J. K. , Mohanty A. K. , Malakar D.
Published: December 2013

Primordial germ cells (PGCs) generated from embryonic stem (ES) cells in different species may be an alternative approach to dealing with the worldwide problem of increasing female infertility. Reprogramming of fibroblasts into induced pluripotent stem cells has been achieved by overexpression of different transcription factors. Here, we report the generation of female goat germ cells from goat induced pluripotent stems cells (giPSC). Goat induced pluripotent stem cells (giPSC) were produced by transduction of adult female goat fibroblast cells with Oct4, Sox2, and Nanog lentiviral particles and further sub-cultured on fibroblast feeder layers. GiPSC were characterised by different methods. These iPSC were found to express alkaline phosphatase, SSEA1, SSEA4, Tra-1–81, and Tra-1–60 surface markers. However, SSEA3 was not observed in giPSC. GiPSC also expressed Oct4, Nanog, and Sox2. Along with Oct4, Nanog, and Sox2, the expression of different transcription factors such as Cdx1, Dapp5, Dax1, Ecat, Eras, Fgf4, Gata6, Lin28, Rex1, and Utf1 was confirmed by RT-PCR. GiPSC were in vitro differentiated and three germ layers were characterised by immunostaining of Gata4 for endoderm, α-Actinin for mesoderm, and β-III tubulin for ectoderm and RT-PCR analysis of GATA4, α-Actinin and BMP4. IPSCs were directed differentiated into germ cells using retinoic acid and bone morphogenetic protein 4 without the inactivation of exogenous factors as these are also required for germ cells development. Differentiated germ cells were characterised by immunostaining against VASA and Dazl proteins. RT–PCR assay was performed for Dazl, Nanog, Nanos1, PUM8, SCP3, Stella, and VASA genes expression. Quantitative PCR was also performed for detection of VASA and Dazl expression during the course of germ cell differentiation. Flow-cytometric analysis of differentiated germ cells was confirmed the presence of germ cells in population of differentiated giPSC. Oocytes/ova-like structures, which were comparable to natural goat oocytes, were observed under scanning electron microscope (SEM). Cumulus–oocyte complex like structure was observed, which was further used for SEM. The study concluded that adult female goat fibroblast cells can be reprogrammed into induced pluripotent stem cells using ectopic expression of Oct4, Nanog, and Sox2 genes and the germ-cells-like cells generated from reprogrammed giPSC could be differentiated into goat oocytes/ova-like structure which have immense applications in human and animal reproduction.
203 ESTABLISHMENT OF AN IN VITRO SCREENING ASSAY FOR DEVELOPMENTAL TOXICITY USING MOUSE EMBRYOID BODIES
Authors: Choi Y. , Jeung E. B.
Published: December 2013

Toxicity is generally referred to as the degree to which a substance can damage an organism. According to numerous reports, determination of toxicity through an in vitro system is an economical and ethical method, compared with an in vivo system using animals. The hES cells can differentiate into three germ layers and the unique feature of hES cells from the three germ layers via the EB formation adds good insight in understanding human developmental biology in vitro. Evaluation of developmental toxicity of a compound on early embryo states can therefore be based on its effect on EB formation. In this study, we used mouse embryonic stem cells (mESC) to investigate the effects of several developmental toxic chemicals on the formation of EBs. We used the EB hanging drop method and tested five toxic chemicals; cytosine arabinoside, dexamethasone, hydroxyurea, indomethacin, 5-fluorouracil, and two negative controls; ascorbic acid and penicillin G. We demonstrated a significant reduction of EB size after treatment with a high dose of each chemical. We evaluated cell toxicity by performing measurements of cell viability after treatment with each chemical. Expression of apoptosis-related genes (p53, Sirt1, p21, Puma, Noxa, Mdm2) and pluripotency marker genes (Oct4, Nanog, Sox2, ZFP206) and differentiation marker genes, such as endoderm (HNF4, AFP), mesoderm (T-brachy), and ectoderm (Pax6) were determined by quantitative real-time PCR. The chemicals induced abnormal differentiation and apoptosis. We confirmed apoptotic positive cells by TUNEL assay. In addition, we also demonstrate the reduction of the size of EBs through expression of apoptosis-related marker genes (p53, Caspase-3, PARP) and necrosis-related marker gene (HMGB1). The results obtained demonstrate that EBs can be used as an in vitro model for testing developmental toxicity.
211 REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY AS A TOOL TO ELUCIDATE THE BIOLOGICAL ACTIVITY OF COMMERCIAL EQUINE CHORIONIC GONADOTROPIN (eCG)
Authors: Alvarez R. H. , Almeida B. E. , Ribela M. T. C. P. , Natal F. L. N. , Melo A. J. F. , Bartolini P.
Published: December 2013

Superovulation in ruminants can be induced with a single injection of equine chorionic gonadotropin (eCG). However, ovarian response is sometimes lower than expected because of, among other factors, the source of eCG used. This study aimed at establishing the physical-chemical profile of commercial eCG, in order to find differences which can be related to their biological activity. Four different commercial eCG products for veterinary use (A, B, C, D) and one eCG chemical reagent from Sigma (St. Louis, MO, USA), here used as reference preparation, were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C4-Grace Vydac 214 TP 54-column (25 cm × 4.6 mm I.D.), with UV detection at 220 nm. All eCG preparations presented at least three peaks with retention times (tR) of ~27(I), 34(II), and 36(III) minutes, with a peak at tR = 27 min common to A, C, D, and Sigma eCG, whereas preparation B did not present this peak. A bioassay test was carried with all of these preparations. Immature 21- to 25-day-old Wistar female rats received the equivalent to 10 IU of eCG of each one of these preparations. Autopsy was performed 48 h later and ovaries were removed and weighed. The average ovarian weight for preparations A, C, D, and Sigma were ~0.0795 ± 0.0107 g, whereas preparation B was 0.035 ± 0.007 g (P < 0.01). Preparation B was not different from saline (0.034 ± 0.002 g). In order to establish which one of these three peaks presented the highest biological activity, a mass equivalent to 10 IU of eCG from peaks I, II, and III of Sigma and of product A were studied. The average ovarian weight of animals injected with material from peak II and III (~0.0285 ± 0.003 g) were similar to that of the control whereas peak I produced ovarian weights of 0.059 ± 0.007 g and 0.075 ± 0.010 g for Sigma and product A, respectively (P < 0.01). These results suggest that the lack of ovarian response to eCG treatments can be related to differences in the physical-chemical profile of commercial eCG products and that RP-HPLC is a fast and reliable tool for detecting these differences.
Supported by FAPESP (Grant 11/13096-0).
220 LENGTH OF DONOR DNA HOMOLOGY TO FACILITATE BI-ALLELIC GENE TARGETING DURING TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE-MEDIATED GENE TARGETING
Authors: Beaton B. P. , Lee K. , Kim J.-H. , Prather R. S. , Wells K. D.
Published: December 2013

Gene editing techniques are increasing in their availability and ease of use. These techniques are an attractive alternative to conventional gene targeting methods via homologous recombination due to the ease of use and the efficiency of targeting. We have previously produced CMP-N-acetylneuraminic acid hydroxylase (CMAH) knockout (KO) pigs in a Minnesota miniature pig genetic background. These pigs were generated using zinc-finger nucleases (ZFN) in combination with donor DNA containing a total length of homology of 1600 bp (800-bp homology on each arm). A second KO was desired in the CMAH –/– genetic background, GGTA1. We used this opportunity to evaluate the effect of total donor homology on transcription activator-like effector nuclease (TALEN) mediated bi-allelic modification. Donor DNA was designed to symmetrically flank the predicted TALEN cleavage site in GGTA1 and was varied in length of total homology (40, 100, 200, 460, 890, and 1800 bp). The TALEN constructs and donor DNA were introduced into fibroblast cells by a lipid-based transfection method. Each donor DNA was co-transfected with a pair of TALEN constructs. In addition, the TALEN pair alone and the longest donor DNA alone were separately transfected to evaluate error-prone non-homologous end joining (NHEJ) repair or background HR, respectively. Five days post transfection, cells were evaluated for the ablation of the α-1,3 gal epitope, which results from bi-allelic disruption of GGTA1. Lectin IB-4 (FITC labeled) was used to detect the presence/absence of the epitope. If a bi-allelic modification to the GGTA1 gene was accomplished, specific binding of the lectin would not occur; therefore, a lack of FITC was evaluated. Donor DNA alone did not result in detectable bi-allelic conversion of GGTA1. As the length of donor DNA increased, the bi-allelic disruption of GGTA1 increased from 0.5% (no donor DNA) to a maximum of 3% (1800 bp). Inclusion of homologous donor DNA in TALEN transfections facilitates HR that competes with error-free NHEJ. The use of donor DNA with TALEN therefore can provide for increased bi-allelic disruption and predictability in the sequence of the resulting mutation.
24 IMPROVEMENT OF PORCINE CLONED EMBRYONIC STEM-LIKE CELLS DERIVATION BY ZONA-MEDIATED EMBRYO AGGREGATION
Authors: Lee D. K. , Park C.-H. , Jeong Y.-I. , Hwang J. Y. , Oh J.-N. , Son D. , Lee C.-K.
Published: December 2013

Porcine embryonic stem cells (ESC) have become an important model for therapeutic cloning using embryonic stem cells derived by somatic cell nuclear transfer (SCNT). However, embryo quality and blastocyst formation have been major limitations for derivation of cloned embryonic stem-like cells. In this study, we tried to overcome these problems by treating with histone deacetylase inhibitors (HDACi) and aggregating porcine embryos. A porcine embryonic fibroblast (PEF) cell line was used as the source of donor cells injected into the enucleated oocytes. First, to confirm the effect of HDACi in cloned embryo quality, cloned embryos were treated with Scriptaid (histone deacetylase inhibitor). The Scriptaid-treated blastocysts (n = 26) showed significantly increased total cell number (29.50 ± 2.10; P < 0.05) than nontreated blastocysts (n = 21; 22.29 ± 1.50). Then, the cloned embryo quality and blastocyst formation were analyzed in aggregates. Three zona-free reconstructed 4-cell stage SCNT embryos were injected into empty zonae from hatched parthenogenic blastocysts. The blastocyst formation and total cell number of cloned blastocysts was significantly elevated for all the aggregates (76.3% and 83.18 ± 8.33 cells/blastocyst) compared with nonaggregated (31.0%, and 27.11 ± 1.67 cells/blastocyst; P < 0.05). Finally, aggregated blastocysts were cultured on a feeder layer to examine the efficiency of porcine embryonic stem-like cells derivation. Aggregated blastocyst showed higher primary colony formation percentage than nonaggregated cloned blastocysts (20.0 ± 12.3% v. 2.2 ± 1.35%, respectively; P < 0.05). In conclusion, the aggregation of pig SCNT embryos at the 4-cell stage could be a useful technique for improving the development rate and quality of cloned pig blastocyst and derivation efficiency of cloned embryonic stem-like cells.
25 PORCINE OOCYTES SELECTION USING BRILLIANT CRESYL BLUE AND EMBRYO DEVELOPMENT AFTER SOMATIC CELL NUCLEAR TRANSFER
Authors: Park E. J. , Song K. Y. , Moon J. H. , Lee B. C.
Published: December 2013

The efficiency of animal cloning through somatic cell nuclear transfer (SCNT) technology is affected by numerous factors such as oocyte quality and donor cell type. Among the factors, oocyte quality can be enhanced by identification and selection of developmentally competent oocytes before in vitro maturation (IVM). Morphological criteria following homogeneous ooplasm, more than 3 layers of cumulus cells, and oocyte diameter have been used. However, the criteria vary between examiners and even within individual. In contrast, use of Brilliant cresyl blue (BCB), a marker of oocyte growing status related to activity of glucose-6-phosphate dehydrogenase (G6PD), can be a more objective selection method for the detection of fully grown oocytes in bovine, equine and porcine. To our knowledge, BCB has been used to select oocytes in parthenogenesis, intracytoplasmic sperm injection (ICSI), and IVF, excluding SCNT in porcine. The aim of this study was to investigate whether oocytes selected by BCB have better ability to develop into blastocysts than oocytes selected by morphological criteria. After aspirated cumulus–oocyte complexes (COC) from porcine ovaries were washed in HEPES-buffered TCM-199 2 times, the COC were selected through morphological criteria and divided randomly into 2 groups. Group 1 (control; 304 COC) was directly transferred into IVM medium and Group 2 was incubated in TALP supplemented with 26 μM BCB for 90 min at 38.5°C in air. Then, COC of the second group were washed twice in TALP, and COC displaying a blue-coloured ooplasm (BCB+) were selected. The 342 BCB+ COC also were transferred into IVM medium and cultured with 5% CO2 in air at 38.5°C, with hormonal supplementation for 22 h and without the hormones for another 22 h. After denudation, the rate of degenerated oocytes and maturation rate were determined. The matured oocytes were used for SCNT and the development and total cell number of blastocysts were observed. Each experiment was repeated at least 3 times. Data were analysed by unpaired Student's t-test using Graphpad Prism (GraphPad, San Diego, CA, USA). No difference was observed between Groups 1 and 2 in the rate of degenerated oocytes (9.13 ± 0.47% and 10.68 ± 2.73%, respectively), metaphase II rate (94.60 ± 2.30% and 89.26 ± 3.76%, respectively), cleavage rate (77.02 ± 1.56% and 80.05 ± 2.31%, respectively), or blastocyst formation rate (9.74 ± 1.91% and 10.74 ± 1.30%, respectively). However, total cell number of blastocyst showed significant difference between Groups 1 and 2 (57.67 ± 1.76 and 77.50 ± 1.50, respectively; P < 0.01). In conclusion, selection of oocytes through BCB staining does not improve their developmental ability with respect to cleavage and blastocyst formation rate, but enhances embryo quality by means of increased total cell number per blastocyst in porcine SCNT.
This study was supported by IPET (#311011-05-2-SB010), MKE (#10033839-2012-21) and TS Corporation.
26 IN VIVO EXOGENIC ORGAN GENERATION WITH ORGANOGENESIS-DISABLED CLONED PIGS AS A PLATFORM
Authors: Matsunari H. , Nakano K. , Kanai T. , Matsuda T. , Maehara M. , Watanabe M. , Umeyama K. , Nagaya M. , Nakauchi H. , Nagashima H.
Published: December 2013

The generation of organs from pluripotent stem cells (PSC) is one of the ultimate goals of regenerative medicine. We have demonstrated that functional organs can be generated in vivo from xenogenic PSC in the body of organogenesis-disabled mice using blastocyst complementation. To apply this principle in generating human organs, a technical platform using large non-rodent mammals is essential. The aim of the present study was to establish a blastocyst complementation system using cloned pig embryos. We generated transgenic-cloned pigs with an apancreatic phenotype via the overexpression of Hes1 (hairy and enhancer of split-1) under the Pdx1 promoter (pancreatic and duodenal homeobox-1). Cloned embryos of apancreatic pigs (host embryos, male) were complemented (i.e. chimerized) by blastomeres of cloned embryos (donor cells, female) with normal developmental competence. Chimeric embryos were cultured for 1 or 2 days before being transferred into the uteri of oestrus-synchronized gilts. The complementation of 292 Pdx1-Hes1 cloned embryos gave rise to 260 (89.0%) blastocysts. The transfer of these blastocysts resulted in 5 male chimeric pigs. Chimerism was confirmed by the detection of host embryo-derived Pdx1-Hes1 and marker transgenes of the donor cells, such as humanized Kusabira-Orange (huKO) or Pdx1-Venus. Chimeric pigs possessed normally formed pancreata entirely derived from the exogenous donor cells. We thus established a blastocyst complementation system in the pig using cloned embryos that would otherwise give rise to apancreatic animals. Chimeric pigs obtained developed normally, maintaining normal serum glucose concentrations up to maturity, and became fertile boars. Mating the chimeric boars with 7 wild-type sows gave rise to 72 fetuses/piglets of which 37 (51.4%) exhibited the apancreatic phenotype. These results indicate that a missing organ can be generated from exogenous cells when functionally normal pluripotent cells chimerize with a cloned dysorganogenetic embryo. Blastocyst complementation using cloned porcine embryos may permit the use of a large animal for the generation of functional organs from xenogenic PSC, including human iPSC. The chimeric boar produced by blastocyst complementation sired fetuses/offspring with the apancreatic phenotype in a Mendelian fashion. Porcine fetuses with an organogenesis-disabled phenotype may provide a useful platform for organ regeneration research.

Table 1.Production of chimeric pigs by complementation and of Pdx1-Hes1 cloned embryos
27 SOMATIC CELL NUCLEAR TRANSFER CLONING AND EMBRYO AGGREGATION IN PIGS
Authors: Buemo C. P. , Gambini A. , Hiriart I. , Salamone D.
Published: December 2013

Somatic cell nuclear transfer (SCNT) derived blastocysts have lower cell number than IVF-derived blastocysts and their in vivo counterparts. The aim of this study was to improve the blastocyst rates and quality of SCNT blastocysts by the aggregation of genetically identical free zona pellucida (ZP) porcine clones. Cumulus–oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the ZP using a protease and then enucleated by micromanipulation; staining was performed with Hoechst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80 V for 30 μs). All reconstituted embryos (RE) were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% PVA) by a DC pulse of 1.2 kV cm–1 for 80 μs. Then, the oocytes were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of free ZP embryos was achieved in a system of well of wells in 100 μL of medium, placing 3 activated oocytes per microwell (aggregation embryo), whereas the control group was cultivated with equal drops without microwells. Embryos were cultivated at 39°C in 5% O2, 5% CO2 for 7 days in SOF medium with a supplement of 10% fetal bovine serum on the fifth day. The RE were placed in microwells. Two experimental groups were used, control group (not added 1X) and 3 RE per microwell (3X). At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality and evaluate if the embryo aggregation improves it. Results demonstrated that aggregation improves in vitro embryo development rates until blastocyst stage and indicated that blastocysts rates calculated over total number of oocytes do not differ between groups (Table 1). Embryo aggregation improves cleavage per oocyte and cleavage per microwell rates, presenting statistical significant differences and increasing the probabilities of higher embryo development generation until the blastocyst stage with better quality and higher diameter.

Table 1.Somatic cell nuclear transfer cloning and embryo aggregation
33 PRODUCTION OF A CLONED FOAL USING MITOCHONDRIAL DNA-IDENTICAL OOCYTES
Authors: Choi Y. H. , Ritthaler J. , Hinrichs K.
Published: December 2013

Recently we reported the birth of a viable foal produced by nuclear transfer (NT) using oocytes recovered from immature follicles of live mares by transvaginal ultrasound-guided aspiration (TVA; 2013 Theriogenology 79, 791–796). This procedure opens the door for production of mitochondrial DNA-identical cloned foals; typically, use of heteroplastic oocytes results in cloned offspring that have different mitochondrial DNA from that of the donor. We selected 2 mares (BL and SM) from the maternal line of the donor, a 23-year old stallion. Genetic analysis confirmed that the mares’ mitochondrial genotype was identical to that of the donor. Oocytes were obtained from the mares by TVA of all follicles ≥5 mm diameter, and were matured in vitro for 20 to 26 h. Donor fibroblasts were treated with 15 μM roscovitine for 24 h, then were directly injected into enucleated oocytes using a Piezo drill. Reconstructed oocytes were activated with 5 μM ionomycin for 4 min followed by injection with sperm extract, then incubation in 2 mM 6-dimethylaminopurine for 4 h. Oocytes from mare SM were assigned to treatment with either Scriptaid (500 nM) or Scriptaid plus vitamin C (50 μg mL–1) for 14 to 16 h, starting at the onset of 6-dimethylaminopurine exposure; mare BL did not provide sufficient oocytes for treatment grouping. Presumptive zygotes were cultured in vitro for 7 to 11 days and blastocysts were shipped for transfer to recipient mares, 1 embryo per mare. In mare BL, 10 aspiration sessions were conducted, 78 follicles were aspirated and 45 oocytes were collected, of which 4 were degenerating. After in vitro maturation, 12/40 (30%) oocytes were mature. Five of 12 oocytes lysed during manipulation; the remaining 7 were cultured and 1 blastocyst (14%) was obtained, which did not yield a pregnancy. In mare SM, 3 aspiration sessions were conducted and 53 oocytes were recovered from 81 follicles. After in vitro maturation, 31/53 (58%) were mature. Four oocytes were lysed during manipulation, 27 were cultured, and 4 blastocysts (15%) were produced, 2 from scriptaid treatment and 2 from scriptaid plus vitamin C. Transfer of these blastocysts yielded one pregnancy (scriptaid treatment); the mare delivered a healthy foal at 328 days of gestation. These results indicate that NT can be successful using low numbers of immature oocytes from selected mares. However, the individual mare may greatly affect the outcome in terms of oocyte number and quality; in this case, mare BL not only yielded fewer oocytes per aspiration session (4.5 v. 17.7 for mare SM; P < 0.001, t-test), but also fewer reconstructed oocytes per oocyte recovered (7/45 v. 27/53, respectively; P < 0.001, Fisher's exact test). Efficiency (14 to 15% blastocysts per reconstructed oocyte cultured; 1 foal from 5 embryos transferred) was similar to that achieved previously in our laboratory using heteroplastic oocytes.
This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, by Kit Knotts, and by Jack Waggoner.
42 EFFECTS OF TRICHOSTATIN A TREATMENT ON GENE EXPRESSION OF CLONED MOUSE 2-CELL AND BLASTOCYST STAGE EMBRYOS
Authors: Marjani S. L. , Carter M. G. , Sung L-Y. , Inoue K. , Rodriguez-Zas S. , Wang L. , Yu H. , Shen H. , Cheng T. , Yang X. , Tian X. C.
Published: December 2013

Trichostatin A (TSA) is a potent inhibitor of histone deacetylases and has been shown to improve cloned embryo pre-implantation and term development. We examined the effects of TSA treatment on cloned mouse embryonic gene expression using microarrays. Cloned mouse embryos were generated using long-term haematopoietic stem cells (LT-HSC) and terminally differentiated granulocytes (Gr-1) as nuclear donors, which have been shown to have significantly different cloning efficiencies (Sung et al. 2006 Nat. Gen. 38, 1323–1328). Late 2-cell and blastocyst stage cloned embryos and control, BDF1 in vivo and IVF embryos (n = 10 from each embryo type and stage, except LT-HSC blastocysts, where n = 5) were snap frozen in liquid nitrogen. Total RNA was isolated from individual embryos and amplified using the TargetAmp 2 round Aminoallyl aRNA amplification kit (Epicentre Biotechnologies, Madison, WI, USA). Amplified RNA from each embryo and a standard reference was labelled with Cy3 or Cy5 and hybridized to the mouse exonic evidence based oligonucleotide (MEEBO) microarray allowing for the interrogation of ~25 000 genes. After Loess normalization, ANOVA with false discovery rate (P < 0.001) was used to identify differentially expressed (DE) genes. A subset of the DE genes was verified by RT-qPCR. These two cell types drastically differed in their potential to give rise to morula/blastocyst stage embryos: LT-HSC: 4.1% v. Gr-1: 38.9%. When treated with 10 nM TSA (Sigma, St. Louis, MO, USA) for 10 h immediately after activation, the morula/blastocyst rate increased to 66.1% for the LT-HSC cloned embryos and to 69.3% for the Gr-1 cloned embryos. At the 2-cell stage, we identified 2172 DE genes between the TSA-treated and untreated LT-HSC embryos. There were 512 DE genes between the Gr-1 and Gr-1 TSA embryos. Interestingly, the cloned embryos were more similar to the in vivo and IVF embryos after TSA treatment at the 2-cell stage, as evidenced by hierarchical clustering and the reduced number of DE genes: LT-HSC v. in vivo = 2622 genes; LT-HSC TSA v. in vivo = 473; Gr-1 v. in vivo = 1448; Gr-1 TSA v. in vivo = 312. By the blastocyst stage, the effect of TSA was considerably less pronounced with 18 and 17 DE genes between the LT-HSC/TSA and Gr-1/TSA embryos, respectively. These data indicate that TSA treatment normalizes 2-cell cloned embryo gene expression, enabling significantly more embryos to develop to the blastocyst stage. Our findings demonstrate that TSA exerted the greatest effect on the LT-HSC embryos, which were the most difficult to reprogram by SCNT.
43 WARMING TEMPERATURE AFFECTS THE VIABILITY AND MEIOTIC COMPETENCE OF IMMATURE PORCINE OOCYTES VITRIFIED IN A CHEMICALLY DEFINED SOLUTION
Authors: Takahashi D. , Funahashi H.
Published: December 2013

The aim of this study was to examine the viability and meiotic competence of porcine oocytes when immature porcine cumulus-oocyte complexes (COC) were pretreated for vitrification at different temperatures (25 and 39°C), vitrified in a chemically defined solution, and warmed at different temperatures (39 and 60°C). Cumulus-oocyte complexes were aspirated from middle-size follicles (3–6 mm in diameter) of abattoir-derived porcine ovaries. After collection, the COC were pretreated with cryoprotectants at different temperatures (25 and 39°C) and vitrified in a serum-free chemically defined solution containing 0.6 mg mL–1 of hydroxypropyl cellulose, basically according to a commercial protocol (Cryotop, Kitazato BioPharma Co. Ltd., Fuji, Japan). The vitrified COC were warmed in 1 M trehalose solution at 39 for 60 s or at 60°C for 30 s. The COC were cultured for in vitro maturation (IVM) in modified porcine oocyte medium (POM) supplemented with 50 μM β-mercaptoethanol, 10 IU mL–1 of eCG, 10 IU mL–1 of hCG, and 1 mM dibutyryl cyclic AMP (dbcAMP) for 20 h and then in the fresh medium without hormonal supplements and dbcAMP for another 24 h. Viability of COC was evaluated under fluorescent microscopy after stain with fluorescein diacetate and propidium iodide. Nuclear maturation of the oocytes was evaluated after 44 h of IVM. Statistical analyses of results from 5 replicated trials were performed by ANOVA with a Bonferroni/Dunn post-hoc test (significance, P < 0.05). Although viabilities of vitrified oocytes after 44 h of IVM [6.0% (9/149) to 37.8% (59/155)] were significantly lower than fresh controls [98.8% (158/160)], the viabilities of vitrified oocytes warmed at 60°C [32.0% (49/160) to 37.8% (59/155)] were significantly higher than those warmed at 39°C [6.0% (9/149) to 10.0% (16/160)]. Maturation rates in vitrified oocytes [2.7% (4/149) to 19.8% (31/155)] were also significantly lower than fresh controls [74.8% (120/160)]. Regardless of temperature during pretreatment for vitrification (25 and 39°C), maturation rate of the oocytes warmed at 60°C after vitrification [16.4% (25/154) to 19.8% (31/155)] was significantly higher than that warmed at 39°C [3.1% (5/160) to 2.7% (4/149)]. In conclusion, these results demonstrate that warming at 60°C for 30 s maintains the viability and meiotic competence of immature porcine COC.
45 EFFECT OF VITRIFICATION ON KIDDING OF CAPRINE EMBRYOS
Authors: Toishibekov M. M. , Blackburn H. , Valieva G. A. , Askarov S. M. , Molzhigitov B. B.
Published: December 2013

This work evaluated different methods: vitrification (V) and super-cooling ultra-rapid vitrification (SCURV). The goat morulae were cryopreserved into the High Security Vitrification (HSV) Kit (Cryo Bio System, Paris, France). The vitrification method was applied according to the method described by Vajta et al. (1998). Both treatments used a vitrification solution [VS; 20% (3.6 mol L–1) ethylene glycol (EG), 20% (2.4 mol L–1) dimethyl sulfoxide (Me2SO), and 0.5 mol L–1 of sucrose in Dulbecco's PBS (DPBS) with 10% BSA in both methods]. In our experiment, we used the Vit-Master™ apparatus (MTG GmbH, Bruckberg, Germany). The supercooled LN facilitates heat transmission between LN and the cryosolution interface and this is efficient for bovine semen and blastocyst cryoconservation (Arav et al. 2002). By surgical flushing of 30 superstimulated (1200 IU of Folligon, Intervet International, Boxmeer, the Netherlands) goats, 137 transferable morulae were harvested; 41 morulae were transferred fresh to synchronized recipients (control) and the others were cryopreserved by V (n = 47) or SCURV (n = 49), respectively thawed or warmed, and transferred to recipients. Embryos were vitrified using the HSV Kit. They were first incubated in 50% VS for 2 min and then transferred for 30 s into 100% VS. Each embryo was loaded by HSV Kit, which was immediately submerged into and stored in LN. Warming was done by placing the narrow end of the straw into DPBS + 0.25 M sucrose for 5 min. Embryos were then transferred into DPBS + 0.125 M sucrose for 3 min and finally to DPBS until transfer. The SCURV morulae were then exposed to 50 and 100% VS at 37°C for 2 min and 30 s, respectively. Embryos after saturation in VS were transferred by HSV Kit and using negative pressure of LN in the chamber for freezing with the VIT-Master. Thawing vitrified embryos was accomplished by placing the vitrified embryos in solutions of sucrose 0.25 and 0.125 M, with exposures of 2 and 3 min, accordingly. After thawing embryos, only good-quality embryos were transferred. The kidding rate following transfer of fresh, frozen-thawed vitrification, and SCURV methods were 25, 17, and 19 kids, respectively. No statistical difference was found for the percentage of does kidding following transfer of thawed embryos after vitrification (36.2 ± 4.4%a) and SCURV methods (38.7 ± 6.5%b). The survival rate following transfer of fresh embryos (60.9 ± 5.3c) was higher and in line with previous findings using VS. Differences were statistically significant (ac, bc: P < 0.05). Importantly, our data suggest that the SCURV method can be used for cryopreservation of goat morulae as the vitrification method. Although further work on the developmental competence of embryos cryopreserved with the SCURV method are needed, these data suggest that with SCURV, a faster freeze rate and lower level of cryoprotectants is able to minimize ice crystal formation and should be further evaluated as a routine mechanism for cryopreserving goat embryos.
66 RELATIONSHIP BETWEEN PLASMA CHLORIDE LEVELS AND FOLLICLE DIAMETER IN CYCLING SPANISH PUREBRED MARES
Authors: Satué K. , Gardon J. C. , Montesinos P.
Published: December 2013

Several studies in cows have shown that plasma levels of chloride are positively correlated with the diameter of the preovulatory follicle (FD), establishing at the same time a significant positive association between plasma levels and intraoviducal concentration of this electrolyte (Hugentobler et al. 2007). These relationships may reflect the influence of changes in systemic metabolic plasma on follicular fluid, physiological events that might be closely related to the quality of the oocyte and granulosa cells of the preovulatory follicle (LeRoy et al. 2004). However, these mechanisms remain unknown in the mare. The objective of this research was to investigate the relationship between plasma chloride concentrations and growth of FD during the ovulatory period in the Spanish purebred mares. This research was approved by the Animal Ethics Committee of the CEU-Cardenal Herrera University. A total of 30 reproductive cycling Spanish purebred mares, aged 4 to 17 years old, were studied. Plasma concentrations of chlorine and follicular growth (FD) were determined daily by extracting blood samples and monitoring of ovarian ultrasound all mares, from Day –5 to the day of ovulation. Plasma chloride concentrations were evaluated by an analyzer with selective electrodes Vetlyte® (IDEXX, Hoofddorp, the Netherlands). To analyse the effect of cycle day on FD and concentrations of chloride, ANOVA was used. The interrelations between both parameters were expressed by Pearson correlation coefficient. The FD increased significantly, from 27.02 ± 2.113 to 40.23 ± 1.377 cm, during the 5 days prior to ovulation. Plasma chloride decreased from 105.6 ± 0.928 mmol L–1 on Day –5 to 104.5 ± 0.128 mmol L–1 on Day –1, and increased significantly, to 107.4 ± 1.442 mmol L–1, on the day of ovulation. No significant correlations between FD and chloride concentrations (r = 0.21) were found. Unlike what happens in the cow, plasma chloride levels affect preovulatory follicle development in Spanish purebred mares to a lesser extent. Nevertheless, plasma chloride or any other electrolytes have some kind of involvement in the dynamics of growth and maturation of oocytes.
75 DETECTION OF EMBRYONIC DEATH BY MONITORING OVARIAN STEROIDS BALANCE AND ULTRASONOGRAPHY IN JAPANESE BLACK CATTLE
Authors: Matsuda H. , Hashiyada Y. , Yamanouchi T. , Imai K.
Published: December 2013

In beef cattle, 20 to 44% embryonic loss occurs during the early stages of pregnancy (Humblot et al. 2001). However, the mechanism of early and late embryonic death is not clear. We investigated occurrence of embryonic death by monitoring ovarian hormones dynamics and checking for the presence of the conceptus after artificial insemination or embryo transfer in beef cattle. Twenty Japanese black females were inseminated (AI) and 12 females were transferred with 1 embryo (ET). Blood samples were collected on Days 21, 24, 28, 38, 48, 58, and 68 post-oestrus (oestrus = Day 0) and then stored at –30°C. Progesterone (P4) and oestradiol (E2) plasma concentrations were analysed using a chemiluminescent immunoassay. On each day of blood sampling, ovaries and presence of a conceptus in the uterus were monitored by ultrasonography. At Day 24 post-oestrus, the presence of the fetus was detected in 8 females (AI, n = 6; ET, n = 2), whereas only 3 females were confirmed to be pregnant at Day 28 and Day 38 post-oestrus. No embryo loss was seen at later stages of pregnancy (Days 48, 58, and 68 post-oestrus). At Day 21 post-oestrus, the conceptus could not be detected by ultrasonography but the E2/P4 ratio provided indication on the pregnancy status of the females that were classified as pregnant (n = 8) or not pregnant (n = 24) at day 24 post-oestrus (1.7 ± 2.2 versus 28.0 ± 34.2 respectively; mean ± s.d.). In the nonpregnant females compared with the pregnant ones at Day 24 post-oestrus, P4 declined below 1 ng mL–1 (0.6 ± 0.2 ng mL–1 v. 8.6 ± 3.9 ng mL–1), whereas E2 blood level remained stable (15.7 ± 21.9 v. 18.1 ± 1.1 pg mL–1). The decrease in P4 levels led to an increase in E2/P4 ratio (1.3 to 37.3 on Day 24). Our study suggests that a large proportion of embryo loss (75%) occurs before Day 24, whereas an additional 16% loss was seen between Day 24 and Day 28 post-oestrus. This embryo loss was shown to be associated with the altered balance of ovarian hormones.