bluuebunny/crossref_metadata_2025 huggingface dataset:
256 EFFECT OF EPIDERMAL GROWTH FACTOR ON IN VITRO MATURATION OF CYNOMOLGUS MONKEY (MACACA FASCICULARIS) OOCYTES
Authors: Yamasaki J. , Okahara-Narita J. , Iwatani C. , Tsuchiya H. , Nakamura S. , Sakuragawa N. , Torii R.
Published: January 2008

Collected oocytes include not only mature oocytes (metaphase II: MII), but also immature oocytes (germinal vesicle: GV, and metaphase I: MI). To establish a dependable artificial indoor breeding program in cynomolgus monkeys, we are planning to carry out in vitro maturation (IVM) using GV and MI oocytes. In this study, we attempted to determine whether different types of feeder layers and epidermal growth factor (EGF) were effective for IVM. Cumulus–oocyte complexes (COCs) were collected from ovaries of 4–10-year-old female cynomolgus monkeys stimulated by the combination of FSH (25 IU kg–1 × 9 days) and hCG (400 IU kg–1) (Torii 2000 Primates 39, 399–406). Oocytes were classified by morphological features: oocytes retaining an intact germinal vesicle nucleus (GV); oocytes that had undergone germinal vesicle breakdown without polar body formation (MI); and oocytes with a first polar body (MII). GV and MI oocytes were co-cultured on monkey cumulus cells (MCC), monkey follicular ovarian cells (MFOC), monkey oviductal cells (MOC), or human solubilized amnion product (HSAP), with TCM-199+10% fetal bovine serum containing epidermal growth factor (EGF; 10 ng mL–1 or 20 ng mL–1). The maturation rate from GV to MII oocytes was 6.7% (MCC), 18.0% (MFOC), 35.7% (MOC), and 28.6% (HSAP) (Table 1). Although higher maturity was observed in MOC and HSAP, the effect of EGF was not found in co-cultures using any feeder layers. The maturation rate from MI to MII oocytes was 33.3% (MCC), 27.8% (MFOC), 55.6% (MOC), and 44.0% (HSAP) (Table 1). The highest maturation rate from GV and MI was observed in co-cultures using MOC. The maturation rate from MI to MII oocytes in the presence of 10 ng mL–1 EGF was 75.0% (MCC) and 73.7% (HSAP) (Table 1), whereas the rate in the presence of 20 ng mL–1 EGF was 59.1% (MCC), 64.3% (MFOC), 92.3% (MOC), and 60.0% (HSAP) (Table 1). Thus, the best maturation rate was a co-culture using MOC as a feeder layer with 20 ng mL–1 EGF. According to our results, maturation rate during IVM depends on the cellular type of feeder layers and the concentration of EGF. EGF is especially effective for maturity from MI to MII oocytes, but not from GV to MI or MII oocytes. Thus, IVM should be carred out under optimal culture conditions, including suitable feeder layer and media plus supplements. In the future, it is important that intracytoplasmic sperm injection be carried out using in vitro-matured MII oocytes for establishment of an artificial indoor breeding program in cynomolgus monkeys.

Table 1. Number of matured oocytes co-cultured with different feeder layers and EGF
260 EFFECTIVENESS OF A COMMERCIALLY AVAILABLE POST-THAW BOVINE SEMEN SEXING KIT IN BOTH MULTIPLE-AND SINGLE-OVULATING COWS
Authors: Curry E. , Pratt S. L. , Lapin D. , Gibbons J. R.
Published: January 2008

Currently, there is no inexpensive method for commercially separating X- and Y-bearing bovine sperm cells. Because dairy heifer calves are significantly more valuable than bull calves, a reliable method of swaying the bovine sex ratio in favor of females is desirable in the dairy industry. The objective of these experiments was to determine the efficacy of a commercially available post-thaw semen sexing kit, HeiferPlus (Emlab Genetics, Arcola, IL, USA), which claims to sway the bovine sex ratio in favor of female calves. Three trials included the insemination of hyperstimulated cows with control or HeiferPlus (HP)-treated semen, nonsurgical embryo collection on Day 7, and a novel combined PCR/Southern blot assay to determine sex. Overall, 271 embryos were collected and a sex was assigned to 265 of them. Chi-square analysis showed that the control group produced a significantly higher proportion (P < 0.005) of female embryos than the HP group (65.0% and 43.0%, respectively). There was no difference in the proportion of transferable versus degenerate embryos, and ANOVA showed no difference in the number of ovulations, embryos, and unfertilized oocytes collected in the control versus the HP groups. Another trial involved the artificial insemination of cows synchronized via OvSynch� (ABS Global, Deforest, WI, USA) followed by fetal sexing via ultrasonography between Days 55 and 58. Of the 152 cows inseminated, 51.3% were pregnant at Day 35. Of the cows inseminated with HP-treated semen, 54.5% became pregnant and 48.0% of the controls were pregnant. HP-treated cows resulted in 54.8% male (23/42) and 45.2% female (19/42) fetuses. Control cows had 52.8% male (19/36) and 47.2% female (17/36) fetuses at 58 days of gestation. Calving records confirmed the ultrasound sexing data and showed that three controls and one HP calf died after 58 days of gestation, and one control and one HP calf died within 2 weeks following birth. Chi-square analysis showed no significant difference in the sex ratio. Semen from six bulls was used in this trial, and pregnancy rates among bulls did not differ (P > 0.05). Computer-assisted semen analysis showed no significant difference in motility or progressive forward motility of control versus HP-treated sperm in any of the bulls. Results of these studies indicated that the HeiferPlus semen sexing kit did not sway the sex ratio in favor of females in either multiple- or single-ovulating cows. In the hyperstimulated cows, the control group actually produced a significantly higher proportion of female embryos than the HP group. No differences in embryo production or embryo grade were observed. Further research is needed to investigate the effects of semen incubation or manipulation on the sex ratio of cattle.
272 BIRTH OF A CALF AFTER INTRACYTOPLASMIC SPERM INJECTION WITH FROZEN - THAWED EPIDIDYMAL SPERM FROM A POSTMORTEM BULL
Authors: Guerrero C. A. , Smith J. , Lynn J. W. , Bondioli K. R. , Godke R. A.
Published: January 2008

The use of postmortem epididymal sperm for intracytoplasmic sperm injection (ICSI) will allow a more effective use of valuable gametes if a breeding male dies unexpectedly. The objective of this study was to determine pronuclear formation and embryo development rates of frozenâthawed bovine epididymal sperm-injected oocytes. Epididymal sperm were harvested by multiple incisions in the cauda epididymides of an abattoir-derived mature, mixed breed beef bull within 5 h postmortem and frozen in 7% glycerol. Oocytes were matured in vitro for 21 h, selected for extrusion of the first polar body, and centrifuged at 6000g to assist in visualizing the microinjection procedure. Oocytes were injected with either frozen–thawed epididymal sperm, frozenâthawed ejaculated sperm (laboratory control), or were sham-injected (control). Piezo-injected oocytes were chemically activated 4 h post-injection in 7% ethanol for 5 min (Treatment A) or exposure to 5 μm ionomycin for 5 min followed by incubation in 10 μg mL–1 of cycloheximide for 5 h (Treatment B). The sperm-injected oocytes were cultured in CR1aa medium from day 0 to day 3 post-injection and then in CR1aa medium supplemented with 5% fetal bovine serum from day 3 to day 8 of in vitro culture. Pronuclear formation was assessed 18 to 20 h after sperm injection. A summary of oocyte activation by treatments indicated that ethanol was more successful than the ionomycin + cycloheximide treatment (Table 1). Cleavage and blastocyst rates were assessed on day 3 and day 8 of culture, respectively. A significantly higher (P ≤ 0.05) fertilization rate was achieved when ejaculated (43%) rather than epididymal (31%) sperm was used in the ICSI procedure. However, this difference in fertilization rate was only noted when ethanol was used for the exogenous activation. Furthermore, the blastocyst rate for epididymal sperm-injected oocytes was significantly greater when using ethanol (14%) compared with ionomycin followed by cycloheximide (4%). The birth of a live bull calf (42.2 kg; 292-day gestation) resulted from the nonsurgical transfer of 2 ethanol-activated Grade 1 day ICSI blastocysts into each of 2 beef recipient females (50%). To our knowledge, this is the first calf produced by piezo ICSI using cryopreserved bovine caudal epididymal sperm. We can conclude
that postmortem epididymal sperm can be collected from genetically valuable males and used for the production of offspring using piezo ICSI.

Table 1. Summary of bovine oocyte activation using ethanol and ionomycin + cycloheximide (Iono + Cyclo) treatments
277 ESTABLISHMENT OF EMBRYONIC STEM CELL LINES DERIVED FROM A EGFP-TRANSGENIC MOUSE AND THEIR SURVIVAL IN THE COCHLEA OF C57BL/6 MICE
Authors: Ahn K. S. , Jeon S. J. , Jung J. Y. , Choi T. , Choi S. J. , Shim H.
Published: January 2008

Embryonic stem (ES) cells isolated from inner cell mass cells of blastocyst-stage embryos are capable of differentiating into various cell lineages. Transplantation of these cells may potentially be a treatment for many degenerative diseases. Such cell therapy has often been tested using allografts of ES cells in mice. However, it has been difficult to locate transplanted ES cells and to avoid the rejection of allogeneic ES cells by the host. The aims of this study were to establish ES cell lines ubiquitously expressing enhanced green fluorescent protein (EGFP) and to test survival of ES cells in allografts into the cochlea of inbred C57BL/6 mice. Nine hatched blastocysts collected from a C57BL/6-green mouse that ubiquitously expresses transgene EGFP were plated onto an inactivated STO feeder layer. Two putative ES-like colonies were obtained from the plated blastocysts, and repeated subculture of these colonies produced two cell lines expressing EGFP. The cell lines possessed typical characteristics of ES cells, including densely packed colonies of the cells with prominent nucleoli, a high nuclear-cytoplasmic ratio, and high alkaline phosphatase activity. In suspension culture, these cells formed simple and cystic embryoid bodies. Undifferentiated EGFP-transgenic ES cells (106 cells per mouse) were injected into the cochlea of five C57BL/6 mice deafened by gentamycin treatment. Although no behavioral changes were noticed until four weeks after the transplantation, histological study revealed that grafted cells survived in the scala media of all injected mice. Incorporation of the cells expressing EGFP into the host was found along the auditory nerve fibers close to the organ of Corti. Such incorporation was also discovered in the area of the spiral ganglion neurons, cochlear sensory epithelia, and stria vascularis. Morphology and size of the cells varied depending on their sites of incorporation. The results from the present study demonstrate that, due to their survival in transplantation without allogeneic rejection as well as ubiquitous and stable expression of EGFP, ES cells from an EGFP-transgenic mouse may be a useful means of studying cell therapy.
279 COMPARISON OF DIFFERENT METHODS OF ISOLATION OF EMBRYONIC STEM CELL-LIKE CELLS FROM IN VITRO-PRODUCED GOAT BLASTOCYSTS
Authors: De A. K. , Malakar D. , Akshey Y. S.
Published: January 2008

The present study was carried out to compare different isolation procedures for embryonic stem (ES) cell-like cells from in vitro-produced goat blastocysts. Goat oocytes were aspirated from slaughterhouse-derived ovaries; matured in vitro in maturation medium containing TCM-199 (HEPES modified) with 5 µg mL–1 FSH, 10 µg mL–1 LH, 1 µg mL–1 estradiol-17β, BSA, and 10% estrous goat serum; and fertilized by freshly collected buck semen. The fertilized oocytes were cultured in embryo development medium containing TCM-199 (HEPES modified), 50 µg mL–1 sodium pyruvate, 3.5 µg mL–1 L-glutamine, gentamicin, essential amino acids, nonessential amino acids, BSA, and 10% fetal calf serum, along with goat oviductal cells for further development. A total of 250 blastocysts and 50 hatched blastocysts were used to isolate and culture goat ES cell-like cells. Inner cell mass cells (ICMs) were isolated mechanically from 100 blastocysts and 20 hatched blastocysts and enzymatically from 50 blastocysts and 15 hatched blastocysts. The ICMs were cultured on 10 µg mL–1 mitomycin-C-inactivated goat fetal fibroblast feeder layer. Primary colony formation was significantly higher (P < 0.01) when hatched blastocysts were used for ICM isolation (28/35; 80%) than when blastocysts were used (77/150; 51.3%). However, when ICMs were isolated mechanically, the percentage of primary colony formation was significantly higher (P < 0.01) for blastocysts (66/100; 66%) as well as for hatched blastocysts (18/20; 90%) than when ICMs were isolated enzymatically (15/50; 30%, and 11/15; 73%), respectively, for blastocysts and hatched blastocysts. ES cell-like cells were also isolated from intact blastocysts and intact hatched blastocysts. But the primary colony formation (27/100; 27%), and (6/15; 40%), respectively, was significantly low (P < 0.01) compared to that for mechanically isolated ICMs. Statistical analysis was done by chi-squire test. When the putative ES cell-like cells were subcultured enzymatically (0.25% trypsin–EDTA), they differentiated after 3 passages. However, when the mechanical subculture procedure was followed, the ES cell-like cells maintained their pluripotent state up to the 9th passage, and three goat ES cell-like cell lines were produced (gES-1, gES-2, and gES-3). The putative ES cell-like cells were stained with alkaline phosphatase substrate solution (Sigma, St. Louis, MO, USA). The RT-PCR mediated expression of Oct-4 was done by a special kit Cells-to-cDNA II (Ambion, Austin, TX, USA). The ES cell-like cells expressed both alkaline phosphatase and Oct-4. It may be concluded that mechanical isolation of hatched goat blastocysts is the best method for isolation of goat embryonic stem cell-like cells, and they are positive for alkaline phosphatase and Oct-4.
28 HIGH HYDROSTATIC PRESSURE IMPROVED DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES FOR HANDMADE CLONING
Authors: Du Y. , Lin L. , Pribenszky C. , Molnár M. , Kragh P. M. , Li J. , Zhang X. , Yang H. , Pedersen A. M. , Villemoes K. , Bolund L. , Vajta G.
Published: January 2008

High hydrostatic pressure (HHP) has been introduced into the field of embryology recently, with the possible mechanism that a sublethal HHP could induce the synthesis of molecular chaperons to protect the embryos from further stresses. Improved cryotolerance has been achieved successfully in HHP-treated mouse (Pribenszky 2005 Anim. Reprod. Sci. 87, 143–150) and bovine (Pribenszky 2005 Reprod. Domest. Anim. 40, 338) embryos, and the semen of bull (Pribenszky 2007 Reprod. Fertil. Dev. 19, 181–182) and boar (Pribenszky 2005 Reprod. Fertil. Dev. 18, 162–163). The objective of the present study was to apply this new technique to in vitro-matured (IVM) porcine oocytes and further investigate its effect in the procedure of handmade cloning (HMC). After 40 h IVM, cumulus–oocyte complexes (COCs) were loaded in 0.5-mL straws by a 2-mL syringe, with HEPES-buffered TCM199 as the loading medium. COCs were then treated with 20 MPa (200 times greater than atmospheric pressure) for 60 min by a pressurizing device (Cryo-Innovation Inc., Budapest, Hungary), with an interval of 120 min between HHP treatment and subsequent HMC. Two different cell lines (from Day 40 fetuses of Yucatan and Danish Landrace breeds (LW1-2)) were used as donor cells for nuclear transfer. A total of 592 reconstructed embryos were produced from both HHP-treated and control groups and were in vitro cultured for 6 days to evaluate the developmental competence through to blastocyst formation. The effect of donor cells on blastocyst development was also investigated. SPSS 11.0 program (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis; values with P < 0.05 were regarded as significant. Blastocyst rates of the different groups are shown in Table 1. Our results indicated that COCs treated with HHP had a much higher blastocyst rate than those untreated (P < 0.01) and this improvement was not affected by using different donor cells for nuclear transfer. In conclusion, the sublethal HHP treatment could improve the in vitro developmental competence of porcine IVM oocytes when they are used for HMC. Further in vivo experiments are required to investigate the long-term effect of HHP on embryo development.

Table 1. Day 6 blastocyst rates of HHP-treated and control groups with different donor cells for nuclear transfer

The authors thank Ruth Kristensen and Janne Adamsen for their help and excellent technical assistance.
286 OSTEOGENIC DIFFERENTIATION IN VITRO OF PORCINE ADULT MESENCHYMAL STEM CELLS
Authors: Lima A. , Monaco E. , Wilson S. , Kim D. , Feltrin C. , Lane S. , Bionaz M. , Hurley W. L. , Wheeler M. B.
Published: January 2008

The quantity and accessibility of subcutaneous adipose tissue in humans make it an attractive alternative to bone marrow as a source of adult stem cells for therapeutic purposes. However, before such a cell source substitution can be proposed, the properties of stem cells derived from adipose (ADSCs) and bone marrow (MSCs) and their differentiated progeny must be compared in an animal model that adequately simulates the structure and physiology of humans. The objective of this work was to induce adult porcine stem cells isolated from subcutaneous adipose tissue and bone marrow to differentiate in vitro along the osteoblastic lineage and to compare their morphological, phenotypic, and genotypic properties. MSCs and ADSCs were isolated respectively from femurs and subcutaneous adipose tissue of adult pigs and cultured in vitro using DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin G-streptomycin, and 5.6 mg L–1 amphotericin B. After 3 passages, cells were differentiated along the osteogenic lineage using lineage-specific inducing medium. Osteogenic medium contained 100 nm dexamethasone, 10 mm β-glycerophosphate, and 0.005 mm ascorbic acid-2-phosphate. Osteogenic cultures were incubated for 4 weeks in 95% air and 5% CO2 at 39�C. Spent medium was replaced with fresh medium every 3 days. Histological staining with alkaline phosphatase, Von Kossa, and alizarin red S were performed at 0, 2, 4, 7, 14, 21, and 28 days of differentiation (dd). At the same time points, RNA was extracted. qPCR was performed on COL1A1, BGLAP, SPARC, and SPP1. As internal control, the geometrical mean of GTF2H, NUBP, and PPP2C was used. Relative mRNA abundance between cell types was calculated using 1/efficiencydCT. The osteogenic differentiation of both MSCs and ADScs was confirmed by the organization of the cells in nodules and by alkaline phosphatase-, Von Kossa-, and alizarin red S-positive staining. The percent relative abundance of the 4 genes in both cell types was COL1A1 (ca. 50) > SPARC (ca. 45) > SPP1 (ca. 5) > BGLAP ( < 0.1). Cell types showed similar mRNA abundance for COL1A1 and SPARC while SPP1 and BGLAP were, respectively, 10- and 19-fold higher in MSCs than in ADSCs. All of the genes had the same pattern among tissues during differentiation except for SPP1, which showed a >10-fold increase at 14 v. 0 dd only for MSCs. Adipose-derived stem cells demonstrated a clear osteogenic differentiation and similar expression and pattern of the two osteogenic genes most abundant in MSCs (COL1A1 and SPARC). However, the higher abundance of SPP1 and BGLAP and the different behavior of SPP1 in MSCs suggest a different transcription profile between the two cell types. From these preliminary results, adipose tissue can be a practical alternative source for stem cells in future human clinical applications.
288 THE EXPRESSION PATTERN OF ACETYLATED ALPHA-TUBULIN IS CONSERVED IN PORCINE AND MURINE SPERMATOGONIAL STEM CELLS
Authors: Luo J. , Megee S. , Dobrinski I.
Published: January 2008

During mammalian spermatogenesis, spermatogonial stem cells (SSCs) reside in the stem cell niche on the basement membrane where they undergo self-renewing divisions. Differentiating daughter cells are located progressively more toward the tubular lumen where they ultimately form spermatozoa. The mechanisms responsible for maintenance of SSCs at the basement membrane are unclear. Microtubules consisting of α/β-tubulin heterodimers are associated with many cellular functions. Reversible acetylation of α-tubulin at Lys40 has been implicated in regulating microtubule stability and function. Acetylation of α-tubulin is abundant in stable microtubules but absent from dynamic cellular structures. Deacetylation of α-tubulin is controlled by histone deacetylase 6 which is predominantly expressed in mouse testis. Here, we tested the hypothesis that differential acetylation of α-tubulin might be involved in maintenance of SSCs. Immunohistochemistry for acetylated α-tubulin (Ac-α-Tu) and the spermatogonia specific proteins PGP 9.5, DAZL, and PLZF were used to characterize the expression pattern of Ac-α-Tu in porcine and murine germ cells at different stages of testis development. In immature boar testes, Ac-α-Tu was present exclusively in gonocytes but not in other testicular cells at 1 week of age, and in a subset of spermatogonia at 10 weeks of age. At this age, spermatogonia are migrating to the basement membrane of the seminiferous tubules, and Ac-α-Tu appeared to be polarized toward the basement membrane. In immature mouse testes, Ac-α-Tu was present in germ cells and Sertoli cells at 6 days of age, whereas at 2 weeks of age, Ac-α-Tu expression was stronger in spermatogonia co-expressing PGP 9.5 and in spermatocytes than in Sertoli cells or PGP 9.5-negative spermatogonia. In adult boar and mouse testes, Ac-α-Tu was detected in a few single or paired spermatogonia expressing PGP 9.5 localized on the basement membrane as well as in spermatocytes, spermatids, and spermatozoa. Spermatogonia with high levels of Ac-α-Tu expressed PLZF but did not express DAZL, suggesting that only undifferentiated spermatogonia maintain a high level of Ac-α-Tu. When seminiferous tubules from 1-week-old and adult boar testes were maintained in vitro for 1–2 days, high levels of Ac-α-Tu were detected in single or paired round spermatogonia with a large nucleus, compared to low levels in elongated paired and aligned spermatogonia. The unique expression pattern of Ac-α-Tu in undifferentiated germ cells during postnatal development appears to be conserved in mammalian testes. Since Ac-α-Tu is a component of long-lived stable microtubules and reducing acetylation of α-tubulin enhances cell motility, these results suggest that stabilization of microtubules might contribute to the maintenance of spermatogonial stem cells.
This work was supported by 1R01 RR 17359-05.
289 TARGETING EFFICIENCIES AND CALCIUM-BINDING PROTEIN PROFILES OF TWO MOUSE EMBRYONIC STEM CELL LINES
Authors: Mamo S. , Kobolak J. , Becker S. , Horsch M. , Beckers J. , Dinnyes A.
Published: January 2008

A concerted performance evaluation and molecular characterization of embryonic stem cells (ESC) will enable us to select cell lines more precisely. However, most of the current ESC studies focus on one or the other approach. Our aims were to compare the targeting efficiencies and gene expression profiles of mouse R1 (Nagy et al. 1993 PNAS 90, 8424–8428) and HM-1 (Selfridge et al. 1992 Somat. Cell Mol. Genet. 18, 325–336) ESCs. Cells were cultured on mitomycin C-treated mouse embryonic fibroblast feeder cell layer and grown in standard ESC medium changed daily [high glucose DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 0.1 mm 2-mercaptoethanol (Sigma, St. Louis, MO, USA), fetal bovine serum (15% v/v; HyClone, Logan, UT, USA), 1000 U mL–1 murine-LIF (CHEMICON International, Temecula, CA, USA), and antibiotics (penicillin: 50 U mL–1, streptomycin: 50 µg mL–1 (Sigma)]. Kinase-inactive murine Tyk2 (Tyk2K923E) vector was constructed by mutating the invariant lysine in the ATP-binding site of the kinase domain. From both ESC lines, 3 � 106 cells each were nucleoporated using the Amaxa A23, A24, and A30 programs (Amaxa Biosystems, Cologne, Germany), and the percentages of positive clones after PCR and Southern blot analyses were scored. For expression profiling, total RNA was isolated from aliquots of R1 ESC at passage 13 and HM-1 ESC at passage 23 using RNeasy Midi kit (Qiagen, D�sseldorf, Germany) procedures. Fifteen µg of total RNA each from the contrasting samples were used for reverse transcription, labeled with either Cy3 or Cy5 dye (Amersham, Freiburg, Germany), and hybridized to the cDNA arrays containing over 21 000 sequences. The microarray results of four independent hybridizations were analyzed to identify differentially regulated genes, and four randomly selected genes were verified by real-time PCR analyses. There was no significant difference at program A23 for the percentage of positive clones [(2.5% R1) and (2.4% HM-1)]. However, as the program number increased (A24, A30), the efficiency of R1 ESC improved (4.7% and 6%, respectively), while that of HM-1 ESC worsened (2.4% and 0%), and these differences between the cell lines were significant (P < 0.05). The gene expression profiles revealed a number of calcium-binding proteins that were significantly (P < 0.01) down-regulated in the HM-1 ESCs compared to R1 ESC, and these were verified by using independent analyses of real-time PCR that confirmed the microarray results. Thus, the variations in the targeting efficiency can be partially explained by differences in the calcium-binding protein levels of these ESCs. An earlier study (Fierro and Liano 1996 J. Physiol. 496, 617–625) described the tolerance and buffering capacity of other cell types with higher calcium-binding protein levels.
294 SUPERSTIMULATION IN THE FIRST FOLLICULAR WAVE, WITHOUT THE USE OF ESTRADIOL IN BONSMARA CATTLE
Authors: Carballo Guerrero D. , Tribulo A. , Tribulo R. , Tribulo H. , Bo G. A.
Published: January 2008

Protocols that control follicular wave emergence and ovulation have had a great impact on the application of commercial on-farm embryo transfer because they permit the initiation of superstimulatory treatments at a self-appointed time. However, the most common approach for the synchronization of follicular wave emergence for superstimulation involves the use of estradiol or its esters that are not commercially available in many countries. Therefore, an experiment was designed to evaluate a protocol in which the superstimulation treatment began at the time of emergence of the first follicular wave without the use of estradiol. Bonsmara donors (29 cows and 41 heifers) were randomly allocated to one of two treatment groups. Donor animals in the experimental group (first wave group) received an intravaginal progesterone releasing device (Cue-Mate, Bioniche Animal Health, ON, Canada) along with PGF (0.150 mg D + cloprostenol, Bioprost-D, Biotay, Argentina) at random stages of the estrous cycle. Cue-Mates were removed 10.5 d later and a second PGF was administered at the same time, followed by GnRH (0.050 mg Lecirelina, Biosin-OV, Biotay, Argentina) 36 h later. Ovulation was expected to occur within 30 h after GnRH (day 0). On day 0 (36 h after gonadotropin-releasing hormone) donors received a new Cue-Mate, and superstimulation treatment was initiated with a total dose of 200 to 260 mg (heifers) or 320 mg (cows) NIH-FSH-P1of Folltropin-V in twice daily decreasing doses over 5 d. The PGF was administered with the last two Folltropin-V injections, and Cue-Mate devices were removed with the last Folltropin-V injection. All donors received 12.5 mg pLH (Lutropin-V, Bioniche Animal Health) 24 h after Cue-Mate removal and were AI 12 and 24 h later. Embryos were collected 7 d after pLH treatment. Donors in the Control group received a Cue-Mate and 2 mg of estradiol benzoate (EB; Bioestradiol, Biotay) and 50 mg of progesterone (Lab. Rio de Janeiro, Argentina), and superstimulation treatments were initiated 4 d later with the same dosages used in the first wave group. The PGF administration, Cue-Mate removal, AI, and embryo collections were done as those in the first wave group. Data were analyzed by ANOVA, and results are shown in Table 1. It was not possible to pass the cervix with the collection catheter in two heifers in the control group, and they were excluded from the analysis. There were no significant effects of donor category (cows v. heifers) or treatment on superovulatory response and embryo quality (P > 0.20). In conlusion, superstimulation on a synchronized first follicular wave is as efficacious as superstimulation following synchronization of follicle wave emergence with estradiol benzoate in Bonsmara cattle.

Table 1. Superovulatory response (means ± SEM) in Bonsmara cows and heifers treated with Folltropin-V during the first follicular wave or 4 d after estradiol administration

Bioniche Animal Health, Belleville, ON, Canada.
296 FREQUENT RECTAL PALPATIONS FOLLOWING SUPEROVULATORY TREATMENT AFFECT SEX RATIO OF EMBRYOS RECOVERED FROM HOLSTEIN HEIFERS
Authors: Ideta A. , Hayama K. , Urakawa M. , Aoyagi Y.
Published: January 2008

Skewing the sex ratio of offspring towards males or females is very important for the livestock industry. Many factors, such as maternal stress, have been suggested to affect the sex ratio (Pratt NC et al. 1989 J. Reprod. Fertil. 87, 763–769). In a recent study (Ideta A et al. 2007 J. Reprod. Dev. doi:10.1262/JRD.19035), the proportion of female embryos recovered from superovulated heifers in which ovulation patterns were observed by repeated transrectal ultrasonography tended to be higher than the expected ratio of 50:50 (66.7%, 26/39). To investigate this phenomenon, we repeated the experiment using a larger number of Holstein heifers. The superovulatory treatment began in the midluteal phase of the estrous cycle (days 8 to 10) and consisted of eight decreasing doses of FSH i.m. (total of 28 Armour units, Antrin R-10, Kawasaki-Mitaka, Kanagawa, Japan) for 4 days with treatment twice daily. Doses of 5 mL and 3 mL of a PGF2α analogue (Veterinary Pronalgon F Injection containing 5 mg mL–1 Dinoprost, Pfizer Animal Health, Tokyo, Japan) were administered i.m. to the animals along with the seventh and eighth FSH treatment, respectively. The heifers were divided into two groups. One group, the rectal palpation (RP) group (n = 9), received transrectal ultrasonography with rectal palpation at 4-h intervals from 36 to 76 h after the first PGF2α treatment. The other group, the Control group (n = 8) received no treatment. The heifers were artificially inseminated at 56 and 72 h after the first PGF2α treatment using frozen–thawed semen from one bull. Seven-day embryos were recovered nonsurgically. Grade 1 to 3 embryos (IETS classification) were selected for this study. Male and female embryos were separated using the loop-mediated isothermal amplification procedure (Hirayama H et al. 2004 Theriogenology 62, 887–896). Data were analyzed using ANOVA and chi-square test. The mean number of recovered ova and embryos was 15.7 � 3.8 (RP) and 14.4 � 2.2 (Control). There was no significant difference in the percentages of unfertilized ova (RP; 14.9 %, 21/141 and Control; 11.3% 13/115, P > 0.05), grade 1 embryos (RP; 51.1%, 72/141 and Control; 54.8%, 63/115, P > 0.05) and grade 1 to 3 embryos (RP; 65.2%, 92/141 and Control; 69.6%, 80/115, P > 0.05) between the two groups. The proportion of female grade 1 embryos in the RP group (66.7%, 48/72) was significantly higher than the expected ratio of 50:50 (P < 0.01). The female ratio of grade 1 embryos in the Control group was 50.8% (32/63). Furthermore, the proportion of female grade 1 to 3 embryos in the RP groups (66.3%, 61/92) was significantly higher than the expected ratio of 50:50 (P < 0.005). The female ratio of grade 1 to 3 embryos in the Control group was 51.3% (41/80). Results indicate that frequent ultrasound examinations and rectal palpations following superovulatory treatment may skew the sex ratio of embryos towards females in Holstein heifers.
297 EFFECT OF GONADOTROPIN-RELEASING HORMONE COMPARED WITH ESTRADIOL-17β AT THE BEGINNING OF A SUPERSTIMULATION PROTOCOL ON SUPEROVULATORY RESPONSE AND EMBRYO QUALITY
Authors: Wock J. M. , Lyle L. M. , Hockett M. E.
Published: January 2008

Success of a superstimulation protocol is largely dependent on avoiding the effects of the dominant follicle. The objective of this study was to determine if gonadotropin-releasing hormone (GnRH) would serve as a suitable alternative to the traditional estradiol-17β in a superstimulation protocol when using a CIDR. Dairy cattle (n = 411) were superstimulated using one of two treatment protocols. The GnRH-treated animals received a CIDR on random days of the estrous cycle (day 0), and GnRH was injected i.m. (100 micrograms of Cystorelin) on day 3. Porcine FSH (Folltropin-V) was administered twice daily in decreasing doses on days 5 to 8, and on day 8, CIDR were removed and 25 mg of dinoprost (PGF2α) was administered twice. The Estradiol-treated animals received a CIDR and 4 mg estradiol-17β i.m. on random days of the estrous cycle (day 0). Porcine FSH was administered twice daily in decreasing doses on days 4 to 7, and on day 7, CIDR were removed and 25 mg of PGF2α was administered twice. All animals were artificially inseminated at standing estrus and at 6-h intervals during estrus. Animals were inseminated a minimum of twice, and those with extended estrous periods were inseminated three times. Animals not showing estrus were inseminated once 48 h after CIDR removal. Seven days following estrus, embryos were collected. For data analysis, animals were divided into lactating cows, dry cows, and heifers. Dosage of FSH for each animal varied according to previous response rates; therefore, animals were further subclassified as high FSH (≥300 mg in 15 mL) or low FSH (<300 mg in 15 mL). As there were no heifers on high FSH, data analysis was done as a 5 � 2 factorial ANOVA. Total number of ova/embryos, quality, development, and fertilization rates were recorded. Analysis of data showed no statistical differences (P > 0.05) between Estradiol- and GnRH-treated groups for number ova/embryos recovered (9.8 � 0.58 v. 9.7 � 0.65), IETS-grade 1 and 2 embryos (4.7 � 0.37 v. 4.5 � 0.41), percentage fertilized (54.1 � 2.5 v. 53.2 � 2.9), and percentage grade 1 and 2 embryos (49.2 � 2.5 v. 49.3 � 2.8), respectively. No significant interactions were found between treatment and lactation status or treatment and FSH dose. Data suggest that GnRH in combination with a CiDR is an effective means of synchronizing follicle wave emergence in a superstimulation protocol.
George Seidel, Josh Walker, John Hassler, and Sam Galphin.
30 INHIBITION OF DNA METHYLTRANSFERASE 1 EXPRESSION IN BOVINE FIBROBLAST CELLS FOR NUCLEAR TRANSFER
Authors: Giraldo A. M. , Lynn J. W. , Purpera M. N. , Godke R. A. , Bondioli K. R.
Published: January 2008

The aberrant expression of DNA methyltransferase 1 (DNMT1) in cloned embryos has been implicated as a possible factor in the improper donor genome reprogramming during nuclear transfer (NT). DNMT1 is responsible for maintaining DNA methylation and the subsequent differentiation status of somatic cells. NT utilizing cell fusion introduces the somatic form of DNMT1 (DNMT1s), which is not normally present in preimplantation embryos and could perpetuate the somatic-like methylation patterns observed in early cloned embryos. The objective of this study was to decrease the level of DNMT1s in bovine fibroblasts using siRNA, prior to their use as donor cells. Fetal fibroblasts were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in 5% CO2 at 37�C. The transfection efficiency of different ratios of siRNA concentrations and transfection reagent (µg:µL; 1:3, 1:6, and 1:9) as well as siRNA concentrations (0.5, 1.0, and 1.5 µg) were determined using fluorescein isothiocyanate (FITC)-labeled control siRNA and fluorescence analyzed by flow cytometry. A DNMT1s-specific siRNA was used to transfect cells at 50 and 80% of confluence. A non-silencing siRNA was used as a negative control. The expression patterns of DNMT1s were characterized by Q-PCR using the δδCT method. ANOVA was used to detect differences in transfection efficiency and gene expression. The combination of 1.0 or 1.5 µg siRNA and a 1:6 siRNA to transfection reagent ratio produced the highest transient transfection rates without affecting cell viability. Cells treated at 50% confluence with 1.5 µg of DNMT1s-specific siRNA at a 1:6 ratio showed 80% less DNMT1s mRNA than cells treated with non-silencing siRNA. At 8 h post-transfection (PT) these cells displayed vacuole-like structures within the cytoplasm, stopped dividing, and died approximately 24 h PT. Cells treated at 80% confluence with 1.0 µg DNMT1s-specific siRNA at a 1:6 ratio resulted in 57, 66, 24, 50, 22, 62, 64, and 56% less DNMT1s than control cells at 4, 6, 8, 10, 12, 24, 48, and 72 h PT, respectively, but without the cytotoxic effects observed when cells at 50% of confluence were treated under the same transfection conditions. These data indicate that optimization of cell density, siRNA concentration, and the ratio between siRNA concentration and transfection reagent are required to decrease the levels of DNMT1s without causing deleterious effects to the cells. However, levels of DNMT1s mRNA can be effectively reduced using siRNA; protein analysis is required to determine if reduction of the transcript results in lower levels of the protein. Subsequent use of these cells for NT will provide insight as to how the presence of this enzyme affects reprogramming in early cloned embryos.
This study was supported by Louisiana State University Board of Regents.
305 TRANSGENIC CLONED PIGS EXPRESSING ORANGE FLUORESCENT PROTEIN KUSABIRA-ORANGE
Authors: Matsunari H. , Onodera M. , Tada N. , Mochizuki H. , Haruyama E. , Ogawa B. , Ikeda Y. , Tohi Y. , Nakayama N. , Saito H. , Ueno S. , Kurome M. , Nagashima H.
Published: January 2008

Transgenic (Tg)-cloned pigs expressing fluorescent proteins are very useful in research such as cell or tissue transplantation studies. In this study, we produced Tg-cloned pigs with an orange fluorescent protein, humanized Kusabira-Orange (huKO), and analyzed the characteristics of these pigs. Fetal fibroblast cells transduced with huKO gene by a gene silencing-resistant retroviral vector, pDΔNhuKO (Suzuki et al. 2002 J. Neurochem. 82, 953–960) were used as nuclear donors. Recipient cytoplasts were prepared using oocytes matured in vitro in NCSU23. The nuclear transfer (NT) embryos were transferred into oviducts of estrus-synchronized recipient gilts after culture in PZM-5 for 1 or 2 days. Cloned pigs obtained were analyzed by Southern blotting for the transgene integration. A total of 23 organs and tissues, including brain, eye, internal and reproductive organs, skin, skeletal muscle, and cartilage were examined by fluorescence stereomicroscopy. Cryo- and paraffin-embedded tissue sections were also prepared to examine fluorescence expression. Immunofluorescent staining of brain cryosection and flow cytometry analysis of peripheral blood cells were performed to identify huKO-expressing cells. Transfer of 429 NT embryos into 4 recipients resulted in 18 (4.2%) cloned offspring. Southern blotting analysis of the cloned pigs confirmed transduction of 2 to 17 copies of the huKO gene in each pig. Autopsy was performed in 6 pigs, and orange fluorescence was confirmed in all the tissues and organs examined in each pig. In addition, prominent orange fluorescence was detected in pancreatic islets and renal glomeruli, indicating that these transgenic pigs are ideal for islet cell transplantation studies. Expression of huKO was also detected in neurons, microglia, and astrocytes in the brain, and granulocytes, monocytes, macrophages, lymphocytes, and platelets in the peripheral blood cells, whereas the expression level in red blood cells was lower. Re-cloning was performed using primary culture fibroblast cells established from 2 Tg-cloned pigs. Transfer of 300 re-cloned embryos into 4 recipients resulted in 3 pregnancies. A re-cloned fetus at Day 44 of gestation showed systemic fluorescence. These results demonstrate that the retroviral vector pD?NhuKO is resistant to gene silencing in pigs, that transduction and expression of the huKO gene had no lethal effects on fetal development, and that a Tg-cloned pig expressing orange fluorescence can be produced by NT of fetal fibroblast cells transduced with the huKO gene.
This study was supported by PROBRAIN.
306 GENERATION OF HUMAN A20 GENE-TRANSGENIC PORCINE FETAL FIBROBLASTS FOR SOMATIC CELL NUCLEAR TRANSFER
Authors: Oropeza M. , Petersen B. , Hornen N. , Herrmann D. , Niemann H.
Published: January 2008

The aim of this project was to produce transgenic pigs with improved features in xenotransplantation, by expressing the human A20 gene to modulate the acute vascular rejection (AVR) reaction ocurring after porcine-to-human xenotransplantation. The A20 gene was originally characterized as a tumor necrosis factor-inducible gene in human umbilical vein endothelial cells (Opipari AW et al. 1990 J. Biol. Chem. 25, 14 705–14 708). The gene is both anti-apoptotic and anti-inflammatory in endothelial cells (Ferran C 2006 Transplantation 82(1 Suppl.), S36–S40) and could thus prevent endothelial cell activation leading to AVR. The hA20-expression vector driven by the CAGGS hybrid promoter (chicken β-actin–rabbit β-globin) containing an IRES-neomycin resistance cassette (9.1 kb) was transfected into porcine fetal fibroblasts (PFF) derived from German Landrace porcine fetal explant cultures. Transfection of 3 � 106 cells was accomplished at 450 V and 350 µF with 10 µg of plasmid DNA. Then, G418-resistant cell clones (800 µg mL–1) were screened by PCR with hA20-specific primers for hA20 integration. Eighty clones were A20-positive in PCR screening from 4 rounds of transfection. One cell clone was verified by DNA sequencing and subsequently used as donor cells in somatic cell nuclear transfer. One hundred sixty-nine embryos were transferred to 2 synchronized peripuberal German Landrace gilts, respectively. Ultrasound examination of recipient sows on Day 22 after embryo transfer confirmed established pregnancies in both recipients. One pregnancy was allowed to go to term and 7 healthy piglets were born, whereas the second pregnancy was terminated on Day 70 of pregnancy for detailed expression analysis of the 8 isolated fetuses. Results showed that the A20 vector can be integrated in PFF, and A20-transgenic PFF can be successfully used in somatic cell nuclear transfer to establish pregnancies. Further analysis will focus on the expression levels and patterns in A20-positive cell clones and the biological function in transgenic piglets. Functional assays will be conducted in vitro and in vivo.
We thank Prof. Beyaert of Ghent University, Belgium for providing us with the expression vector pCAGGSEhA20.
309 PRODUCTION OF PIGS TRANSGENIC FOR HUMAN HEMEOXYGENASE-I BY SOMATIC NUCLEAR TRANSFER
Authors: Petersen B. , Lucas-Hahn A. , Herrmann D. , Kues W. A. , Ramackers W. , Bergmann S. , Carnwath J. W. , Winkler M. , Niemann H.
Published: January 2008

The hyperacute rejection after porcine-to-human xenotransplantation can now be reliably overcome either by transgenic expression of complement regulating factors or by knocking out the gene for α1,3-galactosyltransferase in pigs. The next immunological hurdle is the acute vascular rejection (AVR) primarily caused by endothelial cell activation. Human hemeoxygenase-I (hHO-I) is known to have anti-apoptotic and cell-protective properties. Thus, the expression of hHO-I on porcine endothelial cells could have beneficial effects in a xenotransplantation setting to prevent the formation of AVR. Here we describe the first transgenic pigs with functional expression of hHO-I. Fibroblasts were obtained by an ear punch from a decay accelerating factor (DAF)-transgenic female pig and were cultured in vitro (Kues WA et al. 2005 Biol. Reprod. 72, 1020–1028). Cells reaching confluency of 70 to 80% were detached with EDTA/trypsin and subsequently transfected by electroporation at 450V/350 µF with a vector coding for hHO-I driven by the SV40 promoter. Transfected cells were selected for resistance against G418 (800 µg mL–1) for 14 days. Resistant cell clones were screened for integration of the vector by PCR. One positive cell clone that showed strong expression of the transgene, as determined by immunostaining, was selected for somatic nuclear transfer (Hoelker M et al. 2005 Cloning Stem Cells 7, 35–44). In total, 205 reconstructed embryos were transferred to 2 synchronized peripuberal German Landrace gilts, which gave birth to 9 live piglets, all with normal birth weights. PCR and Southern blot analyses revealed that all of the offspring had integrated the vector into their genome. Two transgenic animals were sacrificed for further characterization and ex vivo perfusion experiments. In these animals, Northern blotting showed weak transcription of the transgene in all xenorelevant organs such as heart, kidney, and liver. Immunostaining of the kidneys revealed that expression of the transgene was confined to the endothelial cell layer. These hHO-I-transgenic porcine kidneys were subjected to an ex vivo perfusion assay and survived ex vivo perfusion for 240 min with AB-pooled human blood, whereas perfusion of 2 non-transgenic controls had to be terminated after 60 min due to problems attributed to immunological rejection. Histology revealed that perfused hHO-I-transgenic kidneys exhibited no indication for xenogenic activation of the human coagulation system. These preliminary results show that functional hHO-I can be expressed in transgenic pigs and that transgenic expression of hHO-I might improve long-term survival of porcine xenografts. These results are encouraging and warrant further studies on endothelial cell activation and the biological function of hemeoxygenase-I in the context of xenotransplantation.
This study was funded by the Deutsche Forschungsgemeinschaft Ni 256/22-1, -2, -3.
36 CLONED BEAGLE DOGS DERIVED FROM FETAL FIBROBLASTS BY NUCLEAR TRANSFER
Authors: Hong S. G. , Jang G. , Kim M. K. , Oh H. J. , Park J. E. , Kang S. K. , Kim D. Y. , Lee B. C.
Published: January 2008

Somatic cell nuclear transfer (SCNT) has been successfully performed in various mammals including sheep, cow, pig, and mouse using a variety of somatic cell types as nuclear donors. Several reports of livestock SCNT indicate that fetal fibroblasts are superior to adult fibroblasts as donor cells. In canine SCNT, however, only adult ear fibroblasts have been used as donor cells (Lee et al. 2005 Nature 436, 641; Jang et al. 2007 Theriogenology 67, 941–947). Accordingly, in the present study, we evaluated the ability of canine fetal fibroblasts to support fetal development to term after nuclear transfer. For SCNT, in vivo-matured oocytes flushed (approximately 72 h after ovulation) from the oviducts of six estrus females were used. Donor cells (fetal fibroblasts) were isolated from the fetus of a beagle bitch obtained at 28 days after artificial insemination. Before using fetal fibroblasts as donor cells, sex was determined by SRY gene detection using PCR. Oocytes were enucleated, microinjected with a female fetal fibroblast, fused by electrical stimulation, and activated chemically (Jang et al. 2007). A total of 50 cloned presumptive embryos were transferred (Day 0) into the oviducts of two naturally synchronous recipient bitches. One pregnancy, detected by ultrasonography on Day 23, was maintained to term and two healthy female puppies weighing 250 and 260 g were born by natural delivery on Day 60. They were genotypically identical to the donor cells, and had phenotypically similar black and white coat color patterns. Analysis of their mtDNA distribution showed that mtDNA in the two cloned beagles originated from one of the six oocyte donor dogs. In conclusion, our results demonstrate the potential of using fetal fibroblasts to facilitate nuclear transfer in the dog. The cloned beagle dogs, which had identical nucleus and mitochondrial DNA, will be provided for biomedical research as bioresources.
This study was financially supported by KOSEF (grant # M10625030005-07N250300510) and the Korean MOE, through the BK21 program for Veterinary Science.