bluuebunny/crossref_metadata_2025 huggingface dataset:
280 MULTILINEAGE POTENTIAL OF PORCINE BONE MARROW AND ADIPOSE-DERIVED MESENCHYMAL STEM CELLS IN 3-D ALGINATE HYDROGELS
Authors: Kim D. , Maki A. J. , Kong H.-J. , Monaco E. , Bionaz M. , Hurley W. L. , Wheeler M. B.
Published: December 2008

Adipose tissue presents an appealing alternative to bone marrow as a source of mesenchymal stem cells (MSC). However, in order to enhance cell proliferation and differentiation, 3-dimensional (3-D) culture may be required. A 3-D culture has benefits due to its more in vivo-like environment. Further, to form a functional tissue, a scaffold material is required to ensure proper shape and allow for efficient delivery of nutrients and growth factors. Alginate, a resorbable hydrogel, is a potential injectable scaffold for fat and bone tissue engineering due to its high biocompatibility, gelation with calcium and slow dissolution in a physiologic environment. In the present study, we examined the viability, gene expression and morphology of MSC, isolated from porcine adipose (ADSC) and bone marrow (BMSC), during osteogenic and adipogenic differentiation in a 3D alginate hydrogel environment for 0, 7 and 14 days (d). ADSC and BMSC were infused into alginate hydrogels, which polymerized upon the addition of Ca+2 ions. Both stem cell types were differentiated into osteoblasts using 0.1 μm dexamethasone, 10 mm beta glycerophosphate and 50 μm ascorbic acid, whereas adipocytes were differentiated using 10 μm insulin, 1 μm dexamethasone, and 0.5 mm IBMX. Osteogenic differentiation was confirmed using alkaline phosphatase, Von Kossa, and alizarin red S staining and adipogenic differentiation was confirmed using Oil Red O. Cell viability and proliferation was quantified using the MTT assay. Gene expression was measured using qPCR. The morphology of ADSC and BMSC differentiated toward osteogenic lineages changed with both cell types forming osteogenic nodules over time. The nodules formed by ADSC were larger in diameter than those formed by BMSC. Unlike the osteogenic cells that formed nodules, the ADSC and BMSC differentiated into adipogenic cells showed no significant changes in cell size or aggregation. Gene expression results indicated increased PPARG expression in BMSC with time whereas ADSC showed a peak of expression on day 7 and then decreased. ADSC showed increased (14-fold) PPRG expression when compared with BMSC. ADSC had 160-fold less expression of ALP than BMSC. BMSC showed a 16-fold higher expression level of BGLAP than ADSC. ADSC showed a 15.8% higher expression than BMSC for COL1a1. Both ADSC and BMSC showed similar trends SPARC expression, but BMSC had a 12-fold higher expression of SPP1 than ADSC. In summary, both types of mesenchymal stem cells successfully differentiated into both lineages and maintained viability in the hydrogel over time. In conclusion, alginate is a viable scaffold material for the differentiation of mesenchymal stem cells for tissue engineering applications. These results allow for future studies using the pig as an in vivo fat and bone tissue engineering model.

This research was supported by the Illinois Regenerative Medicine Institute.
283 COMPARATIVE CHARACTERIZATIONS OF PORCINE MESENCHYMAL STEM CELLS AND SKIN-DERIVED CELLS
Authors: Ock S. A. , Jeon B. G. , Kwack D. O. , Rho G. J.
Published: December 2008

Mesenchymal stem cells (MSC) have high potential in regenerative medicine such as skin substitute for a burn healing. The present study compared the characterizations of porcine MSCs (pMSC) derived from bone marrow to the same porcine adult ear and different porcine fetal skin derived cells on morphology of cell growth, alkaline phosphatase (AP) activity, cell surface (CD) markers (CD 29, 45, 90 and 105) and cell cycle by flow cytometer and protein and mRNA expression of Oct3/4, Nanog and Sox2 by immunocytochemistry and RT-PCR (Carlin et al. 2006 Reprod. Biol. Endocrinol. 4, 8) in 1–3 passage. The pMSC were isolated with Ficoll–Paque Plus (Amersham Science, USA) and skin-derived cells were separated with trypsin-EDTA solution treatment of tissue. Basal medium used for culture of pMSC and skin-derived cells was advanced-DMEM supplemented with 10% FBS and 1% penicillin–streptomycin (10 000 IU and 10 000 μg mL–1, Gibco) and culture medium was exchanged every 3 days. Skin-derived cells were observed similar patterns of colony formation and AP expression by AP chromogen kit (BCIP/NBT) (Abcam Inc., MA, USA) to pMSC on plating culture. pMSC and skin-derived cells were found to have a stronger reaction of CD 45– (St. Louis, MO, USA), CD 29+ (BD Bioscience, US), and CD 90+ (BD Bioscience, US). However, CD 105+ (abD Serotec, UK) weakly expressed in pMSC and skin-derived cells were almost negative. The population of cells at S-phase of the cycle in pMSC was significantly (P < 0.05) greater than those in adult ear and fetal-derived skin cells (30.4 ± 5.3 v. 19.1 ± 3.6 and 21.3 ± 1.7, respectively; mean ± SD). Protein of Oct3/4 (goat polyclonal IgG, Santa Cruz, CA, USA), Nanog (goat polyclonal IgG, Santa Cruz, CA, USA) and Sox2 (rabbit polyclonal IgG, Santa Cruz, CA, USA) were observed at both nucleus and cytoplasm in all cell types. However, the pattern of mRNA expression was different among cell types. Nanog and Sox2 expression was the greatest in pMSC and fetal skin cells, respectively. Oct 4 expression should read extremely low in pMSC compared with skin-derived cells, contrary to expectation. Taken together, pMSC and skin-derived cells revealed similar characterization, indicating that pMSC can expect a role of skin substitute.

This work was supported by grant from Biogreen21 (20070301034040), Ministry of Agriculture and Forestry, Republic of Korea.
298 COMPARISON OF THREE DIFFERENT PROTOCOLS FOR SUPERSTIMULATION OF DAIRY CATTLE
Authors: Steel R. G. , Hasler J. F.
Published: December 2008

Traditionally, successful superstimulation of cattle depended on initiating injections of gonadotrophin at mid-cycle, approximately at second follicular wave emergence. This approach limited the convenience of scheduling donors for superstimulation. With the use of intravaginal progesterone-releasing devices and estradiol 17β, superstimulation can be initiated successfully at any time of the estrous cycle. However, because estradiol cannot be legally injected into cattle in an increasing number of countries, the efficacy of GnRH as an estradiol substitute was investigated. A retrospective analysis was performed on data collected in a commercial bovine embryo transfer program over a period of several years. All donors were lactating dairy cows at least two years of age; approximately 75% were comprised of Holstein and the remainder of Jersey, Guernsey, or Brown Swiss breeds. The three treatments employed were (1) Controls injected twice daily for 4 days with a total of 240 to 400 mg of porcine FSH (Folltropin-V, Bioniche Animal Health, Inc.) in decreasing doses starting between day 7 and day 14 of diestrus, with PG (Lutalyse, Pfizer Animal Health) given at the time of FSH injections no. 5 (35 mg) and 6 (25 mg); (2) Estradiol females received a CIDR (Pfizer Animal Health), 5.0 mg estradiol 17β and 100 mg progesterone in oil on random days of the estrous cycle; FSH was initiated 4 days later as described for controls with CIDR removal at the time of FSH injection no. 6; (3) GnRH females received a CIDR on random days of the estrous cycle and 100 μg GnRH on day 1.5 following CIDR insertion; FSH was initiated 60 h after GnRH injection as described for controls with CIDR removal at the time of FSH injection no. 6. All donors were inseminated with one straw of frozen semen 12 and 24 h after the onset of estrus. Embryos were nonsurgically recovered 7 to 8 days after onset of estrus. Only embryos of grades 1 to 3 (IETS classification) were included in the data. Data were analyzed by ANOVA and Tukey’s hsd test was used to distinguish significance among means as shown in Table 1. Estradiol females produced approximately 2 more ova/embryos per procedure than Control and GnRH groups and an average of 0.8 more embryos per female than did the Control group, but there was no difference compared to the GnRH group. Similar to what has been shown in other commercial embryo transfer data sets, nearly 25% of the donors in each group failed to produce at least one good embryo. Clearly, all three treatments resulted in efficacious superstimulation. In light of the legality issues surrounding the use of estradiol, this study shows that GnRH can be used quite successfully to superstimulate dairy cattle at random times of the estrous cycle.

Table 1.Average numbers of ova and embryos recovered from dairy cows superstimulated with three different protocols

We thank G.E. Seidel, Jr. and S.C. Purcell for assistance with statistical analysis.
3 SELECTION OF UNFRAGMENTED-DNA SPERMATOZOA FROM HEAT STRESSED MICE BY FEMALE UTERINE TRACT AND ZONA PELUCIDA BINDING
Authors: Hourcade J. D. , Perez-Crespo M. , Pintado B. , Gutiérrez-Adán A.
Published: December 2008

Physiological bases of the sperm selection processes within the female reproductive tract before they meet and fertilize the oocyte are unknown. The aim of this work was to determine if one of the keys of spermatozoa selection could be DNA integrity. It has been reported that sperm DNA damage does not impair in vitro fertilization (IVF). However, it has been suggested that the zona pelucida (ZP) is able to select spermatozoa with unfragmented DNA (Liu and Baker 2007 Hum. Reprod. 22, 1597–1602). In this work, DNA damage of spermatozoa was artificially induced by scrotal heat treatment (HT) (42°C, 30 min). Twenty-one days after the HT, spermatozoa were recovered from the epididymis caudae of CD1 mice and from the uterine horns near the cervix (Uc), from the uterine horns near the oviducts (Uo), and from the oviducts (Ov) of CD1 females 1–2 h after mating with HT and control males. In each region we determined numbers of spermatozoa, individual motility and sperm DNA integrity by COMET assay (% DNA in tail, tail length, and COMET moment was calculated). Also, females naturally mated either with HT or control males were killed at Day 14 of pregnancy, and number of foetuses and resorptions was recorded. Additionally, IVF was performed with epididymal sperm from HT or control males, Two hours after IVF attached and un-attached spermatozoa to the ZP were recovered and samples were evaluated for sperm motility (CASA), sperm zona-binding, and sperm DNA fragmentation (COMET). Also cleavage rate of fertilized oocytes with sperm from HT or control males was analyzed. One-way ANOVA was used to compare the results form each group. Epididymal sperm count (12*106 and 4.4*106 for control and HT respectively), sperm motility (75 and 21% respectively) and testis weight (133.90 and 68.76 mg, respectively) were significantly reduced after heat treatment (P < 0.001). For the heat treatment, COMET values decreased significantly during the transit from Uc to Uo and from Uo to Ov (Tail DNA: 25.7, 23.5, and 14.4% respectively, P < 0.01; Tail length: 38.4, 29.4, and 11.2 pixels, P < 0.001; COMET Moment: 12.5, 8.5, and 2 respectively, P < 0.001). Heat treatment reduced numbers of foetuses (7 ± 0.5 v. 5 ± 0.49, control and HT group, respectively), but number of resorptions was not altered. Spermatozoa bound per ZP in IVF experiments (55 ± 7 and 13 ± 6, control and HT, respectively) and cleavage rate (61 ± 1 v. 15 ± 6, control and HT, respectively) were significantly reduced in the HT group. Two hours after IVF, spermatozoa attached to the ZP in HT group showed a significant decrease in COMET parameters as in tail length (59.46 ± 2.895 v. 34.66 ± 3.531), and in tail moment compared with unattached spermatozoa. Our results indicate that DNA integrity sperm selection mechanisms are present in both the female tract and the ZP. We suggest that genital tract and sperm-ZP binding process plays an important role in selection of sperm with normal chromatin DNA.
301 INTRACYTOPLASMIC SPERM INJECTION (ICSI) MEDIATED GENE TRANSFER ASSISTED BY ACTIVATION WITH A DOUBLE EXPOSURE TO IONOMYCIN AND 6-DIMETHYLAMINOPURINE OR DEHYDROLEUCODINE
Authors: Bevacqua R. J. , Pereyra-Bonnet F. , Olivera R. , Salamone D. F.
Published: December 2008

ICSI-mediated gene transfer is a powerful technique used to produce transgenic mice and pigs. However, this method of transgenesis has not been applied in bovine due to low embryo development, which is presumed to be a consequence of a failure in sperm factor delivery after ICSI in this species. To bypass this problem, we assisted ICSI with chemical activation, employing two Ionomycin (Io) exposures and 6-Dimethylaminopurine (DMAP) or a novel drug, Dehydroleucodine (DhL). Cumulus–oocyte complexes were aspirated from ovaries obtained from a local slaughterhouse and in vitro matured in bicarbonate-buffered TCM-199 containing 10% FBS, 10 μg mL–1 FSH, 0.3 mm sodium pyruvate, 100 μm cysteamine and 10 UI mL–1 penicillin. IVM conditions were 6% CO2 in humidified air at 39°C for 24 h. MII oocytes were selected and used immediately for ICSI. Sperm samples were frozen/thawed by standard procedures. Coincubation of spermatozoa with DNA construction (pCX-EGFP) was carried out in Na citrate 2.8%, with 0.5 μg plasmid million–1 spermatozoa for 5 min at 0°C. Then, spermatozoa were used for ICSI. Injected oocytes were activated in 5 μm Io for 4 min and placed in TCM-199 for 3 h to allow second polar body emission. Afterwards, some of the oocytes were subjected to a second exposure of Io. Oocytes exposed once or twice to Io were then incubated with 2 mm DMAP (groups Io-DMAP and 2Io-DMAP) or 5 mm DhL (groups Io-DhL and 2Io-DhL) for 3 h. Control groups (Io and 2Io) were not treated with DMAP or DhL. Embryos were cultured in the IVM droplets. EGFP expression was daily evaluated in fluorescence microscope under blue light (488 nm). Significant differences between groups were evaluated by Fisher test (Table 1). DhL chemical activation improved neither development nor transgenesis rates. The double Io exposure significantly improved embryo development. The second exposure to Io previous chemical activation with DMAP resulted in an increase in the percentage of EGFP-expressing embryos. Our results indicate that activation with double Io-DMAP could be considered an alternative assistance for ICSI mediated gene transfer in bovine.

Table 1.Effect of activation assisting transgenic ICSI on development and expression of bovine embryos
31 PORCINE SKIN-DERIVED STEM CELLS MAY BE A SUPERIOR SOURCE OF DONOR NUCLEI FOR EFFICIENT GENETIC MODIFICATION OF CLONED EMBRYOS
Authors: Hao Y. H. , Ross J. W. , Sutovsky P. , Wax D. , Zhong Z. S. , Murphy C. , Rieke A. , Samuel M. , Spate L. , Prather R. S.
Published: December 2008

Somatic cell nuclear transfer (SCNT) in pigs relies primarily on the utilization of fetal-derived fibroblast cells, and the resultant clones tend to exhibit a significant level of phenotypic instability, which may be due to epigenetic reprogramming and/or genomic damage in the donor cells. In addition to the compromised phenotypic stability, production of transgenic clones through SCNT is inefficient, because the restricted lifespan of somatic donor cells in culture can be limiting when the genetic modification requires selection. In contrast, stem cells proliferate rapidly and do not undergo senescence at a high rate, so the selection process can be extended. Since there is no report of an embryonic stem cell line derived in the pig that could contribute to the germ line, we decided to investigate the utility of porcine skin-derived stem cells (SSCs). Porcine SSCs were isolated from the skin on the back of day 35 to 50 Yorkshire fetuses. The SSCs were cultured continually in SSCs medium (DMEM/F12 containing B-27, 20 ng mL–1 of epidermal growth factor, and 40 ng mL–1 of basic fibroblast growth factor) at 37.8°C, 5% CO2, 95% air. The SSCs expressed the neural progenitor marker nestin, as well as genes that are critical for pluripotency, such as Oct4 and Stat3. The SSCs proliferated actively in vitro and retained a normal karyotype after long-term culture. Electron microscopy revealed 2 distinct cell types within the spheres; elongated cells at the sphere periphery had invaginated nuclear envelopes and prominent nucleoli, and these cells displayed few, but large elongated mitochondria with transversal cristae as well as large cisternae of rough endoplasmic reticulum. In contrast, the cells in the center of the spheres were predominantly round-shaped, with a large round nucleus or cuboidal. The SSCs can be genetically modified with long-term positive selection, and 50 μg mL–1 G418 appeared to be an appropriate dose of G418 for selection of the transfected SSCs. Finally, NT embryos reconstructed with SSCs showed high rates of pre- and post-implantation development.The cell number in the blastocyst stage embryos derived from cloning with the SSC was significantly higher than those of the blastocysts derived from IVF (28.5 ± 1.9, 16.8 ± 4.0, respectively, P < 0.05), although there was no significant difference in blastocyst formation rates between these groups (21 to 25%). Three of the animals became pregnant in 4 surrogate gilts which received cloned embryos and reached to term. Two healthy male cloned piglets and 1 healthy female cloned piglet are genetically identical to the SSCs.

Funding for this study was provided by the National Institutes of Health.
36 TREATMENT OF PORCINE FETAL FIBROBLASTS WITH DNA METHYLATION INHIBITORS OR HISTONE DEACETYLATION INHIBITORS IMPROVES DEVELOPMENT OF NUCLEAR TRANSFER EMBRYOS
Authors: Jin D. I. , Kenji N. , Han R. X. , Choi S. M. , Kim M. Y. , Park C. S.
Published: December 2008

Epigenetic status of the genome of a donor nucleus has an important effect on the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). DNA methylation inhibitors [such as 5-aza-2′-deoxyctidine (5-aza-dC), zebularine, and RG108] and histone deacetylase inhibitors [such as trichostatin A (TSA), sodium butyrate (NaBu), and scriptaid (SCR)] have been widely used for the alteration of the levels of the epigenetic modification of somatic cells. This study was designed to investigate the DNA methylation status of porcine fetal fibroblast cells treated with TSA or 5-aza-dC and to determine whether treatments with DNA methylation inhibitors or histone deacetylation inhibitors could improve the in vitro development of porcine reconstructed embryos. When the levels of DNA methylation in the PRE-1 sequence (repeat sequence in a euchromatic region) were examined by bisulfite sequencing following treatment of porcine fetal fibroblast cells with TSA or 5-aza-dC for 1 h, DNA methylation was decreased in 5-nm or 50-nm concentrations even if they were not significantly different. To evaluate the effect of DNA methylation inhibitors and histone deacetylation inhibitors on development of porcine nuclear transfer embryos, porcine fetal fibroblast cells were treated with 5 nm of 5-aza-dC, zebularine, or RG108 for 1 h, or with 50 nm of TSA, NaBu, or SCR for 1 h, or treated with both 50 nm TSA and 5 nm 5-aza-dC for 1 h before NT. The reconstructed embryos were electrically fused and cultured in PZM-3 for 6 days. Developmental rates of the reconstructed embryos from donor cells treated with 5-aza-dC, zebularine, or RG-108 to blastocysts significantly increased compared to the control group (21.4, 23.3, and 22.1 v. 12.3%). Blastocyst rates of the reconstructed embryos from donor cells treated with TSA, SCR, and NaBu also were significantly improved compared to the control group (30.0, 23.9, and 22.4 v. 14.5%), and TSA treatment was the highest in blastocyst rates among the treated groups. However, the development rate to the blastocyst stage was not affected when the combination of TSA and 5-aza-dC was treated. In conclusion, treatment of donor cells with DNA methylation inhibitors or histone deacetylase inhibitors improved the subsequent blastocyst development of porcine reconstructed embryos even though combined treatment with both inhibitors had no beneficial effect.
38 EFFECTS OF 5-AZACYTIDINE ON DNA HYPERMETHYLATION STATUS OF CLONED MINIATURE PIG EMBRYO
Authors: Kim H. S. , Jeong Y. I. , Kim J. H. , Choi J. , Jeong Y. W. , Hossein M. S. , Hyun S. H. , Hwang W. S.
Published: December 2008

During somatic cell nuclear transfer, somatic cells should undergo epigenetic reprogramming in order to attain successful totipotency. Although there are many reasons of low efficiency of cloning, the aberrant DNA methylation status in donor cells is thought to reduce efficiency and increase abnormalities in cloned embryos. The methylation level of cloned embryos is higher than that of in vivo-produced or in vitro-fertilized embryos before occurring de novo methylation. This hyper DNA methylation status has been considered as a reason for the abnormal development of cloned embryos and the related decrease in term number. The aim of this research is to validate the DNA methylation pattern in cloned porcine embryos and to confirm whether reduction of hyper DNA methylation levels results in an improvement in cloning efficiency. First, immunostaining was used to evaluate the stage-specific changing pattern of DNA methylation of cloned embryos. In this result, we demonstrated that the 1-cell stage embryos and blastocyst had the highest level of methylation compared to 2- or 4-cell stage embryos. Second, this methylation level was higher in SCNT-derived embryos compared to parthenogenic embryos, suggesting that the methylation level was associated with the nucleus of donor cells. Third, cloned embryos were treated with various concentration of 5-azacytidine, which is a demethylating agent, to find adequate concentration. In results, 500 nm of 5-azacytidin group from 0 to 24 h after activation produced significantly more blastocysts compared to control group (control; 5.4 ± 2.3, 500 nm/0 24-h treatment; 12.7 ± 2.1, P < 0.05) and also showed low DNA methylation level of inner cell mass different from that of control group. In conclusion, our results suggest that cloned porcine embryos show typical demethylation. But, they are generally on a hyper level of DNA methylation. This high DNA methylation level is associated with somatic cells and affects on blastocyst development. We also suggest that 5-azacytidine could improve blastocyst development rate of cloned porcine embryos by aiding weak and abnormal demethylation process.
40 STREPTOLYSIN O PERMEABILIZED AND MITOTIC EXTRACT TREATED GOAT FETAL FIBROBLASTS FOR USE IN NUCLEAR TRANSFER
Authors: Kish S. L. , Wilson J. A. , Picou A. A. , Lynn J. W. , Godke R. A. , Bondioli K. R.
Published: December 2008

Nuclear transfer (NT) technology has been limited by low rates of embryonic development, high pregnancy loss and low survival of cloned offspring. A major cause is thought to be incomplete or incorrect reprogramming of the somatic cell nucleus. An alternative NT method involves inducing nuclear envelope breakdown (NEBD) and chromatin condensation (CC) with a mitotic cell extract prior to NT. In this study, caprine fetal fibroblasts were permeabilized with streptolysin O (SLO) and NEBD and CC induced with a mitotic extract prior to NT. Reprogramming was indirectly assessed by measuring DNA methylation in early NT embryos. Permeabilization and resealing of fibroblasts were assessed by propidium iodide (PI) uptake and CC was monitored by staining with Hoechst 33342. All data for percent of cells staining was analyzed with one way ANOVA with significance at P ≤ .05. Cells were treated with 0, 150, 300, or 450 U mL–1 SLO for 15, 40, 65, or 90 min. A SLO concentration of 300 U mL–1 for 40 min produced the most efficient permeabilization. Permeabilized cells were treated with extracts from 2, 4 or 6 × 106 mitotic cells for 30, 45 or 60 min. There was no difference in percent of cells with CC between treatments (45–67%) but a higher percentage of treated cells had CC than nontreated cells. Permeabilized and treated cells were resealed by addition of 0, 2.0, 2.5, or 3.0 mm CaCl2 to the culture medium and incubated for 60, 90, or 120 min. Addition of 2.5 mm CaCl2 and incubation for 60 min produced the most efficient resealing. Caprine embryos were produced by NT using oocytes from abattoir and superstimulated ovaries. Fibroblasts were permeabilized by incubation with 300 U mL–1 of SLO at 37°C for 40 min in HBSS with 10 mm dithiothreitol. Fibroblasts were then incubated with mitotic extract for 30 min at 37°C, then cultured in DMEM supplemented with 10% FBS and 2.5 mm CaCl2 for 1 h. Nuclear transfer was performed using untreated donor cells and SLO and mitotic extract treated (SLOT) fibroblasts from the same fetal cell line. All donor cells were at passage 5 or less and contact inhibited by confluence prior to treatment (SLOT) or fusion (NT). NT with untreated cells produced 21 (8%) 2–4 cell and 10 (4%) 4–8 cell embryos. NT with SLOT cells produced 23 (9%) 2–4 cell and 9 (4%) 4–8 cell embryos. In a separate experiment, IVF embryos were produced from abattoir sourced ooctyes for DNA methylation analysis. Embryos were fixed, permeabilized, and immunostained with an antibody against 5-methylcytosine and observed with a confocal laser scanning microscope. Total DNA methylation in individual nuclei of NT and IVF embryos is being determined by image analysis with Image J software.
49 CLONING OF CANCER SNIFFING DOG BY SOMATIC CELL NUCLEAR TRANSFER
Authors: Park J. E. , Oh H. J. , Hong S. G. , Kang J. T. , Kim M. J. , Kim D. Y. , Ra J. C. , Goo J. , Lee B. C.
Published: December 2008

Somatic cell nuclear transfer (SCNT) has been considered as an important method for the production of genetically identical animals and propagation of valuable genotypes. A 3-year-old dog with an outstanding talent for sniffing a cancer was used as a donor dog for SCNT. The donor dog cannot get pregnant by natural fertilization after she suffered pyometra leading to the removal of her reproductive tract. Thus, the aim of this study was to preserve the cancer sniffing dog with excellent genetic background by SCNT. Skin fibroblast from a piece of ear tissue was isolated and cultured. For SCNT, in vivo matured dog oocytes were collected by flushing the oviducts of estrus females. The oocytes were enucleated, microinjected with a donor cell, and fused by electric stimulation. One hundred thirty out of 163, reconstructed embryos (79.8%) were successfully fused. The cloned embryos were chemically activated and transferred into the uterine tube of 7 naturally estrus-synchronized surrogates as with previous report (Lee et al. 2005 Nature). One among them became pregnant, maintained to term, and delivered 4 cloned live offspring (3.1%, live birth/transferred embryos), weighting 450, 410, 430, and 420 g by Caesarean section. The parentage analysis was performed and all cloned pups were genetically identical to the donor dog, and their mitochondrial DNA was originated from their oocyte donor dogs. The present results demonstrated the successful production of four cloned cancer sniffing dogs by SCNT. To date, all cloned puppies have been healthy, growing without apparent abnormalities. Cloned dogs will be trained for cancer sniffing and will be able to provide potential scientific evidence toward the cancer detecting mechanism.

This study was financially supported by KOSEF (grant # M10625030005-08N250300510), SNU foundation (Benefactor; RNL BIO), and the Korean MEST, through the BK21 program for Veterinary Science.
52 EFFECT OF TRICHOSTATIN A ON DEVELOPMENTAL POTENTIAL OF INTER-SPECIES CLONED GAUR (BOS GAURUS) EMBRYOS
Authors: Srirattana K. , Laowtammathron C. , Devahudi R. , Imsoonthornruksa S. , Sangmalee A. , Tunwattana W. , Lorthongpanich C. , Sripunya N. , Keawmungkun K. , Phewsoi W. , Ketudat-Cairns M. , Parnpai R.
Published: December 2008

This study was carried out to investigate the effect of trichostatin A (TSA) treatment on interspecies cloned gaur (Bos gaurus) embryos development and implantation rate after transfer to bovine (Bos taurus) recipients. The bovine (Bos taurus) enucleated oocytes were used as recipient cytoplasm for male and female gaur fibroblasts. After electrical fusion, oocytes were separated into two groups, TSA treatment and control. For the TSA group, the oocytes were placed in EmCare (ICPbio, Ltd., Auckland, New Zealand) holding medium + 50 nm TSA for 1 h. The fused oocytes were activated by 7% ethanol + 50 nm TSA for 5 min at room temperature and 10 μg mL–1 cycloheximide + 1.25 μg mL–1 cytochalasin D + 50 nm TSA at 38.5°C under 5% CO2 in air for 5 h. Then the embryos were cultured in mSOFaa medium + 3 mg mL–1 bovine serum albumin (BSA) + 50 nm TSA up to 10 h. After 10 h, the reconstructed embryos were transferred to embryo culture medium without TSA and culture for 2 days at 38.5°C under 5% CO2, 5% O2, 90% N2. The control embryos were cultured with the same culture system without TSA supplementation. Eight-cell stage embryos were selected and co-cultured with bovine oviductal epithelial cells in culture medium at 38.5°C under 5% CO2 in air for 5 days. Half volume of the culture medium was replaced daily. Two blastocysts at days 7 or 8 derived from male fibroblasts of treated and non-treated TSA were non-surgically transferred to each synchronized estrous bovine recipients. The statistical analysis was done by ANOVA and the comparison of means by Duncan’s Multiple Range Test (DMRT). The development to blastocyst stage was not different among male and female, treated and non-treated TSA embryos which range between 34.8 to 39.3%. The pregnancy rate at 40 days after recipients received cloned embryos derived from male fibroblasts treated v. non-treated TSA was 11% (2/18) v. 10% (1/10) (Table 1). One recipient which received a non-treated embryo gave birth by C-section on March 4, 2008. The male gaur calf died from respiratory problem at 12 h after birth. Eight bovine microsatellite markers analysis confirmed that the newborn gaur was derived from the donor gaur fibroblast. In this study, TSA has no effect on pre-implantation cloned gaur embryos development either derived from male or female gaur fibroblasts. Cloned gaur calves could be produced by interspecies cloning using bovine oocytes as recipient cytoplasm.

Table 1.Pregnancy and birth rates after transferred cloned gaur embryos derived from male fibroblasts to recipients

This study was supported by National Center for Genetic Engineering and Biotechnology (BIOTEC) and Suranaree University of Technology.
62 EXPOSURE TO ETHYLENE GLYCOL AND DIMETHYL SULFOXIDE CAUSES ACTIVATION AND SPINDLE ANOMALIES IN BUFFALO (BUBALUS BUBALIS) OOCYTES
Authors: De Blasi M. , Mariotti E. , Rubessa M. , Di Francesco S. , Campanile G. , Zicarelli L. , Gasparrini B.
Published: December 2008

Despite the increasing interest, buffalo oocyte cryopreservation is still inefficient, especially in terms of blastocyst development after IVF. The aim of this work was to evaluate chromatin and spindle organization of buffalo in vitro-matured oocytes after vitrification/warming by cryotop and after their simple exposure to cryoprotectants (CP). An overall amount of 251 COC was selected and matured in vitro. In the vitrification group, COC were first exposed to 10% ethylene glycol (EG) + 10% DMSO for 3 min, and then to 20% EG + 20% of DMSO and 0.5 m sucrose, loaded on cryotops, and plunged into liquid nitrogen within 25 s. Oocytes were warmed into a 1.25 m sucrose solution for 1 min and then to decreasing concentrations of sucrose (0.625 m, 0.42 m, and 0.31 m) for 30s each. In order to test CP toxicity, COC were simply exposed to the vitrification and warming solutions. Two hours after warming, oocytes were fixed and immunostained for microtubules using a method previously described (Messinger SM and Albertini DF 1991 J. Cell Sci. 100, 289–298), stained for nuclei with Hoechst, and examined by fluorescence microscopy. Fresh in vitro-matured oocytes were fixed and stained as controls. Data were analyzed by chi-square test; results are shown in Table 1. The percentages of MII oocytes in the control and vitrification groups were greater than in the toxicity group, in which a greater percentage of telophase II stage oocytes were found compared with both the control and vitrification groups, indicating occurrence of activation. Of the MII oocytes, both exposure to CP and vitrification procedures gave greater percentages of oocytes with abnormal spindle and abnormal chromatin configuration compared with the control. An unexpected datum was the evidence of a significant percentage of spontaneously activated oocytes in the toxicity group. We speculate that the lack of activation in the vitrification group may be related to the slowing down of metabolic activity subsequent to thermal shock, and hence, that activation after vitrification may occur later than 2 h post-warming. In conclusion, the simple exposure to CP causes activation of the COC and damage to the cytoskeleton similar to that induced by the whole vitrification protocol. The damages to the meiotic spindle and DNA fragmentation may lead to aneuploidy incompatible with subsequent embryo development and account for the poor embryo development currently recorded in buffalo.

Table 1.Chromatin and spindle organization in oocytes vitrified and exposed to cryoprotectants
64 INCREASED REACTIVE OXYGEN SPECIES ACTIVITY IN PORCINE OOCYTES AND ZYGOTES VITRIFIED BY SOLID SURFACE VITRIFICATION
Authors: Gupta M. K. , Uhm S. J. , Park M. R. , Das Z. C. , Lee H. T.
Published: December 2008

This study investigated the effect of reactive oxygen species (ROS) activity on the viability and fertilization rate of porcine IVM oocytes and on the developmental competence of IVF zygotes after solid surface vitrification (SSV). The SSV was performed essentially as we described earlier (Gupta MK et al. 2007 Theriogenology 67, 238–248). Briefly, matured oocytes having first polar body or zygotes having second polar body were equilibrated in TCM-199 medium supplemented with 20% (v/v) fetal bovine serum (FBS), 2% (v/v) dimethyl sulfoxide (DMSO), and 2% (v/v) ethylene glycol (EG) for 10 to 15 min. Equilibrated oocytes/zygotes were then rinsed 3 times (<30 s) in vitrification solution consisting of 17.5% (v/v) EG, 17.5% DMSO, 5% (w/v) polyvinylpyrrolidone, 13.7% (v/v) sucrose, and 20% (v/v) FBS in HEPES-buffered TCM-199 medium and then placed in groups of 10 to 15 oocytes/zygotes per 1- to 2-μL droplet onto aluminum foil kept directly over the liquid nitrogen. Vitrified oocytes/zygotes were then directly plunged in liquid nitrogen. Warming was performed in 2 steps in 0.4 and 0.2 m sucrose solution, each for 5 min. Nonvitrified, untreated oocytes/zygotes were used as controls whereas oocytes/zygotes treated with cryoprotectants only were used as toxicity controls (TC). The viability was evaluated by morphological appearance and fluorescein diacetate staining, and ROS level was assessed by 2′,7′-dichlorofluorescene fluorescence staining. Results showed that the percentages of live oocytes (75.0 ± 1.3%) and zygotes (85.5 ± 1.4%) in the SSV group were significantly lower than those of control (100.0 ± 0.0%, 100.0 ± 0.0%) and TC groups (97.3 ± 2.4%, 96.2 ± 0.6%), respectively. Similarly, the ROS levels were significantly greater in SSV groups than in control groups (P < 0.05). Addition of antioxidant (50 μm β-mercaptoethanol) to vitrification and culture media reduced the level of ROS in both oocyte and zygote groups, but did not improve the percentage of live oocytes (73.4 ± 2.2%) and zygotes (88.1 ± 3.7%) after warming. No beneficial effect of antioxidant was observed on the fertilization rate of vitrified oocytes. However, it did improve the cleavage rates of vitrified oocytes (30.0 ± 0.3% v. 21.3 ± 0.8%) and zygotes (64.2 ± 0.6% v. 54.3 ± 0.7%). These data suggest, therefore, that an increase in ROS activity may be associated with poor developmental competence of vitrified oocytes and embryos.
67 GLUTATHIONE ADDED TO CULTURE MEDIUM AND CRYOPROTECTANT SOLUTION ENHANCE THE VIABILITY OF EMBRYONIC STEM CELLS AFTER THAWING
Authors: Kim G. A. , Lee S. T. , Lee E. J. , Choi J. H. , Lim J. M.
Published: December 2008

We have attempted to improve cell viability of embryonic stem cells (ESC) after freezing and thawing, and, as a result, an efficient cryoprotectant for cryopreserving of mouse ESC was selected in a previous study. This study was subsequently conducted to examine whether the addition of glutathione (GSH), an antioxidant, to the optimized culture medium and cryoprotectant solution could improve post-thaw cell viability of ESC. Embryonic stem cells of a mouse E14 cell line were subcultured 5 times and subsequently employed for the cryopreservation. Dulbecco’s minimal essential medium, to which 100 μm GSH was added or not, was used as a basal medium for ESC culture and a freezing solution. Dimethyl sulfoxide (10%, v/v) and ethylene glycol (10%, v/v) were used as cryoprotectants. In experiment 1, intracytoplasmic concentrations of reactive oxygen species (ROS) in ESCs subcultured and cryopreserved in GSH-free and GSH-containing medium was measured immediately after cryopreservation. A significant (P < 0.0001) decrease in ROS level was detected in ESC cultured and cryopreserved in GSH-containing medium compared with GSH-free medium. Cell viability was subsequently assessed immediately and 72 h after thawing in experiment 2. Improved post-thaw viability was detected when GSH-containing media were employed. There were no significant differences of the viability between fresh and frozen–thawed ESC, regardless of time of assessment immediately (P > 0.1325; 0.653 v. 0.604) and 72 h (P > 0.2998; 0.671 v. 0.626) after treatment. However, there was lower post-thaw viability at 0 h (P < 0.0024; 0.671 v. 0.544) and at 72 h (P < 0.0138; 0.742 v. 0.59) in ESC cultured and cryopreserved in GSH-free medium than that in ESC cultured and cryopreserved with GSH-containing medium. These results demonstrated that use of GSH as an antioxidant affects the level of ROS and promotes post-thaw survival of frozen ESC.
79 CHOLESTEROL QUANTIFICATION DURING CRYOPRESERVATION IN BOAR SPERM SAMPLES ENRICHED IN OR DEPRIVED OF CHOLESTEROL
Authors: Tomás C. , Hernández M. , Mocé E. , Martínez E. , Vá J. M. , Roca J.
Published: December 2008

Sperm membranes suffer significant lipid changes during cryopreservation similar to initial steps in capacitation, in which a reduction in plasma membrane cholesterol (pmCHO) is observed. Methyl-β-cyclodextrin (MBCD) or cyclodextrin pre-loaded with cholesterol (CLC; Purdy PH and Graham JK 2004 Cryobiology 48, 36–45) have been used to decrease or increase the pmCHO, respectively, in different mammalian spermatozoa. In this study, pmCHO levels were assessed during the cryopreservation process in boar sperm samples deprived of (D) or enriched in (E) CHO. Single sperm-rich fractions from 14 boars were divided in 4 aliquots and frozen in 0.5-mL straws after dilution in a lactose-egg yolk extender with a final concentration of 20% egg yolk and 3% glycerol (Control sample, C) and supplemented with MBCD (1 mg/120 × 106 cells; D sample) or CLC at 1 (E-1 sample) or 3 (E-3 sample) mg/120 × 106 cells. The pmCHO level was quantified at 17°C in pre-diluted sperm samples (basal pmCHO) and at 3 steps of the cryopreservation process: after cooling at 5°C (step 1), after the addition of 3% glycerol (step 2), and immediately after thawing (step 3). The pmCHO was quantified by an enzymatic colorimetric test (Spinreact®, Sant Esteve de Bas, Spain) at 520 nm, following the protocol described by Moore AI et al. (2005 Cryobiology 51, 241–249). Data (least squares means ± SEM) were expressed as micrograms of CHO/106 sperm and analyzed as a mixed-model ANOVA. Because there were significant differences (P ≤ 0.05) between ejaculates, this effect was included as random. The level of pmCHO in C, D, and E-1 samples followed the same trend without significant differences (P ≤ 0.05) among them. That level increased (P ≤ 0.05) at step 1 (0.55 ± 0.17; 0.52 ± 0.17; and 0.66 ± 0.17 for C, D, and E-1, respectively), compared with the basal level (0.24 ± 0.02), and decreased (P ≤ 0.05) to the basal level at steps 2 (0.23 ± 0.07; 0.23 ± 0.07; and 0.30 ± 0.07 for C, D, and E-1, respectively), and 3 (0.07 ± 0.02; 0.08 ± 0.02; and 0.13 ± 0.02 for C, D, and E-1, respectively). Although the pattern in E-3 was similar to other treatments, the level of pmCHO was greater (P ≤ 0.05) than those at step 1 (0.75 ± 0.17), 2 (0.51 ± 0.07), and 3 (0.16 ± 0.02). In conclusion, the pre-freezing treatment of sperm samples with methyl-β-cyclodextrin, to reduce the CHO, did not modify the CHO level of plasma membrane of boar spermatozoa. However, treatment with cyclodextrin pre-loaded with CHO at 3 mg/120 × 106 cells increased significantly the CHO level of plasma membrane, which was evident throughout the cryopreservation process.

Supported by AGL2006-07769/GAN, AGL2005-00760/CICYT, Madrid, and GERM (04543/07), Murcia, Spain.
81 MEMBRANE PERMEABILITY CHARACTERISTICS OF BOVINE OOCYTES IN THE PRESENCE OF DIFFERENT CRYOPROTECTANTS
Authors: Wang X. , Al Naib A. , Sun D.-W. , Lonergan P.
Published: December 2008

Development of effective cryopreservation protocols for mammalian oocytes depends on knowledge of fundamental cryobiologic characteristics of the oocyte membrane. The aim of the present work was to study the membrane permeability of immature (germinal vesicle, GV, n = 95) and IVM (metaphase II, MII, n = 104) bovine oocytes to water, dimethyl sulfoxide (Me2SO), or ethylene glycol (EG). Bovine ovaries were obtained from a local abattoir and oocytes were recovered by follicular aspiration. Volumetric changes of bovine oocytes in anisotonic solutions (osmolarity ranges from 130 to 1250 mOsm kg–1) were measured after 20 min of exposure at 24 ± 1°C and the osmotically inactive volume of the oocytes (Vb) was determined using the Boyle van’t Hoff relationship. Volumetric changes of oocytes during exposure to 1.8 m ethylene glycol (EG) or 1.4 m Me2SO at 24 ± 1°C were also measured. Volumetric change was recorded over a 20-min period using an inverted microscope and images were analyzed using the UTHSCSA ImageTool (Version 3.0 University of Texas Health Science Center, San Antonio, TX). The data were fitted to a 2-parameter model to determine the water (Lp) and solute permeability (Ps) of GV and MII stage bovine oocytes using the Runge–Kutta 4 method in Berkeley Madonna 8.3.11 (University of California, CA). Data were analyzed by ANOVA and are expressed as means ± SD. The osmotically inactive volume of GV and MII bovine oocytes were found to be 16.07 and 26.08% of the isotonic cell volume, respectively. In conclusion, these data highlight differences in sensitivity between GV and MII oocytes and provide the fundamental basis for the development of vitrification protocols for bovine oocytes.

Table 1.Bovine oocyte membrane permeability parameters (Lp and Ps) when exposed to 1.8 m EG or 1.4 m Me2SO at 24 ± 1°C
83 CHANGES IN BLASTOCYST LINEAGE DIFFERENTIATION AND NUTRIENT SENSING ARE MEMORIZED IN RESPONSE TO MATERNAL LOW-PROTEIN DIET
Authors: Burt E. , Brooks S. , Cameron I. T. , Fleming T. P. , Eckert J. J.
Published: December 2008

Studies have consistently shown that conditions experienced during the periconceptional period including culture in vitro and maternal diet composition can profoundly alter the development of the embryo/fetus and can result in adult diseases such as obesity, hypertension, and cardiovascular disease. This programming occurs during the pre-implantation period. Here, we have examined blastocyst phenotype changes in response to maternal protein undernutrition during the pre-implantation period and determined whether such adaptations are stable through a change in environment. Embryos were flushed at Day 3.25 to 3.75 from naturally mated MF1 mice fed either a control (NPD) or low-protein diet (LPD) from day of plug. Blastocysts were differentially labeled using the TNBS-anti-DNP-complement method with propidium iodide and Hoechst, and cells were counted on z-series with overlays using Metamorph software (Molecular Devices, Sunnyvale, CA). For outgrowths, blastocysts flushed from LPD or NPD mothers were placed into KSOM supplemented with 10% FCS and amino acids at uterine fluid concentrations (Porter et al. 2003 Pediatr. Res. 53, 46A abst). Outgrowths were cultured for an additional 96 h and scored daily with final 4′,6-diamidino-2-phenylindole (DAPI) nuclei counts. Neither total cell numbers nor lineage divergence was affected by maternal diet until just before implantation (Day 3.75) when blastocysts from LPD mothers had significantly more total cells due to an increase in trophectoderm (TE) compared with NPD blastocysts (ANOVA; Table 1). Upon outgrowth, LPD embryos spread over a greater area although with similar nuclei numbers compared with NPD blastocysts. Moreover, inhibition of the mTOR signaling pathway with rapamycin (rapa) showed the expected dose-responsive decrease of spreading in NPD but not in LPD outgrowths (Table 1). Our data suggest a compensatory upregulation of the TE lineage after mild maternal protein undernutrition. This response becomes evident just before implantation and is, together with altered nutrient sensing and signaling sensitivity, sustained throughout peri-implantation development in vitro regardless of culture environment. Thus, we show that adaptations evident by the blastocyst stage and induced by maternal environment can subsequently be memorized and are likely to contribute to long-term programming of phenotype independent of that environment.

Table 1.Blastocyst and peri-implantation development respond to maternal diet

Funding by DOHaD, Gerald Kerkut Trust, and NICHD are gratefully acknowledged.
92 INDUCTION OF FIRST POSTPARTUM OVULATION, AFTER ADMINISTRATION OF GONADOTROPIN-RELEASING HORMONE OR ESTRADIOL BENZOATE, IN NELLORE COWS
Authors: Pinheiro V. G. , Satrapa R. A. , Simões R. A. L. , Rosa F. S. , Barros C. M.
Published: December 2008

There are reports in the literature indicating that the presence of calf, body condition score, number of births (multiparous v. primiparous), and breed are factors that influence the duration of postpartum anestrus in beef cows. The purpose of the present study was to evaluate, during early postpartum, the time of the reestablishment of LH stocks, measured by the hypothalamic-pituitary axis responsiveness to exogenous administration of gonadotropin-releasing hormone (GnRH) or estradiol benzoate (EB). Primiparous lactating Nellore cows (n = 38, body condition score 2.5 to 3.5, on a 0 to 5 scale) were kept in a Brachiaria brizantha pasture. The animals were randomly allocated into 2 groups, according to hormone treatment: EB group (1 mg EB, i.m., Estrogin®, Farmavet, Sao Paulo, Brazil; n = 20) and GnRH group (100 μg, licerelina, i.m, Gestran Plus®, ARSA S.L.R., Buenos Aires, Argentina; n = 18). In each group, half of the animals were supplemented with a balanced diet based on cotton meal and ground corn. The drugs were administered from 7 days postpartum (±4 days), at intervals of 7 days, until the occurrence of the first ovulation that was observed by weekly ultrasonography (US, Aloka 900, Tokyo, Japan; 7.5-MHz transrectal probe). The data were analyzed by ANOVA (Proc GLM, SAS Institute, Cary, NC). Within the GnRH and EB groups, the first ovulation occurred 15 and 33 days postpartum, respectively, whereas the group averages (± SD) occurred earlier in cows treated with GnRH compared with those treated with EB (43.0 ± 3.8. v. 75.1 ± 8.7 days postpartum, respectively; P < 0.01). In the EB group, 4 animals did not ovulate until Day 140 postpartum. There was no interaction of treatment (GnRH, EB) × nutrition (supplemented, nonsupplemented). When data from the EB and GnRH groups were combined, there was a significant difference between supplemented and nonsupplemented animals (44.4 ± 6.2 v. 68.9 ± 7.3 days, respectively; P < 0.02). The results indicate that GnRH induces ovulation from Day 15 postpartum, suggesting the presence of sufficient LH in the pituitary to induce the first ovulation at this time. However, EB administration did not induce ovulation in this period, possibly because of the sensitivity of the hypothalamus to negative feedback of estrogens, inhibiting the preovulatory LH surge. These results also indicate that nutritional supplementation anticipates the first postpartum ovulation induced by GnRH or EB.

Support by FAPESP(Sao Paulo, Brazil); fellowship from FAPESPA; fellowship from CAPESB.