bluuebunny/crossref_metadata_2025 huggingface dataset:
206 EFFECTS OF A COMBINATION OF A PRID, PGF2α AND eCG, ON ESTRUS SYNCHRONIZATION AND PREGNANCY RATE FOLLOWING EMBRYO TRANSFER IN HOLSTEIN HEIFERS
Authors: Aoyagi Y. , Ideta A. , Matsui M. , Hayama K. , Urakawa M. , Miyamoto A. , Miyake Y.
Published: December 2006

Successful bovine embryo transfer requires synchronization of luteolysis, estrus and ovulation. The objective of the present study was to evaluate the effect of a combination of a PRID, PGF2� and eCG, on estrus synchronization and pregnancy rate in recipient heifers. A PRID� (ASKA Pharmaceutical Co., Ltd., Tokyo, Japan) was inserted into the vagina at random days of the estrous cycle for 7 (n = 35) or 9 (n = 43) days. Two days before removal of the PRID, the heifers were injected with PGF2� IM (2 mL Resipron�-C containing 0.25 mg mL-1 cloprostenol; ASKA). About half of the heifers in each group received 250 IU eCG IM (Serotropin�; ASKA) at the time of PRID removal. Blood was collected several times from the start of treatment for 7 (n = 9) or 9 (n = 9) days and on the day of embryo transfer by jugular venipuncture; plasma was immediately separated and stored at -20�C until assayed for plasma concentrations of estradiol-17α (E2) and progesterone (P4). The E2 and P4 determinations were performed by enzyme immunoassay after extraction by diethyl ether. Pregnancy was determined by ultrasonography on Day 30 (Day 0 = estrus). The rates of successful standing estrus (no. in estrus/PRID inserted), embryo transfer (no. transferred/estrus), and pregnancy (no. pregnancy/transferred) were compared between groups. Data were analyzed by chi-square analysis or Fisher's PLSD test following ANOVA. Injection of eCG at the time of PRID removal had no significant effect on the rates of successful standing estrus, embryo transfer, or pregnancy (P > 0.05). The proportion of heifers treated for 9 days that exhibited standing estrus (93%, 40/43) was significantly higher than the proportion of heifers treated for 7 days that exhibited standing estrus (66%, 23/35, P < 0.01). Of the heifers that were treated for 9 days, the proportion of heifers exhibiting standing estrus within 2 days after the end of treatment was significantly higher (93%, 37/40) than for heifers that were treated for 7 days (65%, 15/23; P < 0.01). Pregnancy rates of heifers treated for 9 days (84%, 32/38) and 7 days (81%, 17/21) were not significantly different. The E2 : P4 ratio normally increases during follicle growth and CL regression. The plasma E2 : P4 ratio between the time of injection of PGF2α and the time of PRID removal was significantly higher for heifers that were treated for 9 days than it was for heifers that were treated for 7 days (P < 0.01). These results suggest that a combination of PRID treatment for 9 days and injection of PGF2α 2 days before PRID removal successfully synchronized estrus in recipient heifers and led to high pregnancy rates following embryo transfer.
208 EFFECT OF TWO CATTLE EMBRYO CO-CULTURE SYSTEMS ON CALVING RATE
Authors: Duszewska A. M. , Wojdan J. , Gawron W. , Wenta-Muchalska E. , Was B. , Wisniewska A. , Chromik A. , Reklewski Z.
Published: December 2006

The objective of this study was to compare the calving rate after transfer of IVM/IVF/IVC embryos co cultured on both Vero and Vero/BRL cells (Duszewska 2000 Theriogenology 54, 1239–1247). Cumulus–oocyte complexes (COCs) were matured in TCM-199 supplemented with 10% FBS, 0.02 IU mL−1 FSH, 1 µg mL−1 17β-estradiol, 0.2 mM Na pyruvate, and 50 µg mL−1 gentamicin for 24 h at 38.5°C in 5% CO2. Spermatozoa were prepared by the swim-up procedure. COCs were fertilized in Fert-TALP supplemented with 6 mg/mL−1 fatty acid-free BSA, 0.2 mM Na pyruvate, 50 µg mL−1 gentamicin sulfate, 20 µM penicillamine, 10 µM hypotaurine, 1 µM epinephrine, and 2 µg/mL−1 heparin for 20 h at 38.5°C in 5% CO2. The zygotes were randomly allocated to one of the co-culture systems: Vero (2 × 103 cells in a 40-µL drop; 20 zygotes per drop), and Vero/BRL (1 × 103 Vero cells and 1 × 103 BRL cells in a 40-µL drop; 20 zygotes per drop). The zygotes from Vero and Vero/BRL were cultured for 168 h post-insemination in drops of Menezo B2 supplemented with 10% FBS until 144 h and from 144 h to 168 h without FBS, at 38.5°C in 5% CO2. Next, the blastocysts (Grade 1, according to IETS Manual) from Vero and Vero/BRL were transferred to recipients. The recipients were monitored daily for heat behavior, examined by ultrasound after 35 days and 65 days, and then observed monthly to confirm pregnancy. The results are presented in Table 1. Statistical significance was tested using the chi-square test. In spite of better development of cattle embryos on Vero/BRL cells than on Vero cells (P < 0.05), a lower rate of calving was obtained after transfer of these embryos to recipients than for those on Vero cells (P < 0.001). Higher loss of pregnancy after transfer of Vero/BRL embryos was observed in Days 35–65, which may indicate early fetal resorption. All calves were born naturally, healthy, and with normal weight.

Table 1.Calving rate after transfer of embryos co-cultured on Vero cells and on Vero/BRL cells

This work was supported by KBN Grant 2P06D05228.
213 THE EFFECT OF eCG TREATMENT IN AN ESTRUS SYNCHRONIZATION PROTOCOL ON PREGNANCY RATES AFTER EMBRYO TRANSFER IN HOLSTEIN HEIFERS
Authors: Okazaki T. , Sasaki E. , Hasegawa K. , Takani T. , Abe S.
Published: December 2006

Recent studies have shown that the presence of accessory or multiple corpora lutea (CL) and increased progesterone (P4) concentrations reduced early embryonic mortality in cattle. The objective of this study was to evaluate the effect of equine chorionic gonadotropin (eCG) treatment on the number of CL, the P4 concentrations, and pregnancy rates after embryo transfer (ET). Holstein heifers (n = 120) from 7 dairy farms received an intravaginal progesterone-releasing device (CIDR; InterAg, Hamilton, New Zealand) and 2 mg IM of estradiol benzoate (EB; Gynandol®; Sankyo, Tokyo, Japan) at random stages of the estrous cycle. After 7 to 9 days, CIDRs were removed and 15 mg of prostaglandin F2α (PG; Pronalgon®; Pfizer Japan, Nagoya, Japan) were administered, followed by 100 µg IM GnRH (Conceral®; Takeda Pharmaceutical Co., Ltd., Osaka, Japan) 2 days later (Day 0). The heifers were placed at random into 3 groups for eCG treatment. The eCG was not administered in a control group (n = 53); heifers in other 2 groups received 1000 IU eCG (Peamex®; Sankyo, Japan) IM at the time (0 h group, n = 37) or 48 h before (48 h group, n = 30) PG injection/CIDR removal. On Day 7, heifers were examined by ultrasonography (Aloka SSD500; Aloka, Tokyo, Japan) for number of CL; heifers with at least one functional CL received an in vivo-derived frozen–thawed embryo by direct transfer. At the same time, a blood sample was collected to determine P4 concentration. Pregnancy rates were determined on Days 30 and 60 by ultrasonography and rectal palpation, respectively. The data were analyzed by ANOVA and means were compared with Fisher's PLSD. Proportional data were analyzed by the chi-square test. P4 concentrations (mean ± SD) on Day 7 were 1.8 ± 1.0, 5.6 ± 3.3, and 2.2 ± 1.1 ng mL−1 for the control, 48 h, and 0 h groups, respectively (48 h vs. control and 0 h; P < 0.001). The number of CL on Day 7 were 1.1 ± 0.4, 2.5 ± 1.4, and 1.8 ± 0.9 for the control, 48 h, and 0 h groups, respectively (control vs. 48 h and 0 h, and 48 h vs. 0 h; P < 0.01). Pregnancy rates did not differ between 0 and 48 h groups but both were higher than in the control group (Table 1). Results suggest that the estrus synchronization protocol with administration of eCG at the time of CIDR removal or 48 h earlier significantly increased the number of CL and the P4 concentration, and improved pregnancy rates in Holstein heifers after ET.

Table 1.Pregnancy rates of Holstein heifers synchronized with CIDR and PG and treated with eCG
218 BREEDING TRIAL OF CONGENITAL HYDROCEPHALUS OF JAPANESE BLACK CATTLE USING EMBRYO TRANSFER
Authors: Shioya Y. , Suzuki T. , Sato Y. , Yoshikawa Y. , Hosokawa Y. , Fukunari K. , Sugimoto Y. , Watanabe T. , Takenouchi N.
Published: December 2006

Hydrocephalus is a congenital defect of cattle. Births of Japanese Black calves with forehead doming were reported in Tohoku District from 1988 to 1989. The common finding on autopsy was the presence of excessive amounts of cerebrospinal fluid with expanding lateral ventricles without evidence of microbiologic infection. Breeding records for pedigree registration have been maintained by the Wagyu Registry Association in this area since 1960. A survey of the pedigrees suggested that an autosomal recessive gene might be responsible for the defect. The incidence of doming calves ceased by an advised mating system which excluded the suspected sires after 1990. Molecular genetics is useful for disclosing DNA sequences of a recessive gene and for detecting the carriers of the gene. This trial was conducted to produce congenital hydrocephalus calves for gene analysis using embryo transfer on a small number of the remaining cows that gave birth to affected calves over 10 years ago. One donor cow, 11 years old, was superovulated with 24 to 25 AU of FSH (Antrin; Kawasaki Mitaka K.K., Kawasaki, Japan) and inseminated with frozen semen of a suspected bull. Collected embryos were transferred on the day of the collection and the rest of them were frozen and transferred later. Superstimulation was repeated 4 times in this cow at an interval of 76-105 days during and 8-month period. A total of 4, 13, 17, and 18 embryos of good quality were collected. Eighteen fresh embryos were transferred into 7 (2 embryos each) and 4 (single embryo) recipients. Frozen embryos were transferred into 6 recipients (single embryo). Seven of 17 recipients became pregnant and gave birth to 8 calves. Three of 6 male calves born and one of 2 females showed symptoms of hydrocephalus. Clinically, 3 of the affected calves had a domed forehead and squint-eyes and were unable to stand. One calf underwent a fatal convulsion and ataxia 29 days after birth. Pathological findings of affected calves showed dilated bilateral ventricles with increased cerebrospinal fluid as typical lesions of hydrocephalus. Embryo transfer was demonstrated to be a useful tool for investigation of a potentially heritable disease. Gene scanning with microsatellite markers using the tissues from the calves and the related cattle was done. The suspected gene was found near the centromere of chromosome 3 (BTA3). It was possible to diagnose the mutant-carrier cattle using two DNA markers, INRA006 (17.0 cm) and BL41 (43.2 cm).
22 RESPIRATORY ACTIVITY AND ULTRASTRUCTURAL FEATURES OF BOVINE SOMATIC NUCLEAR TRANSFER EMBRYOS
Authors: Abe H. , Aoyagi K. , Aoyagi S. , Shiku H. , Matsue T. , Sendai Y. , Hoshi H.
Published: December 2006

We succeeded in determining oxygen consumption of individual bovine embryos non-invasively and quantitatively by scanning electrochemical microscopy (SECM). Recently, we have found that there is a close relationship between high oxygen consumption and developmental ability of bovine IVF embryos. However, the relationship between the respiratory activity and the quality of bovine embryos reconstructed by somatic cell nuclear transfer (SCNT) is still unclear. The aims of this study were: (1) to assess the oxygen consumption of single bovine SCNT and IVF embryos; and (2) to examine the ultrastructural features of SCNT embryos. Bovine oocytes were matured in IVMD101 medium (Research Institute for the Functional Peptides, Yamagata, Japan) and enucleated. The recipient oocytes were activated by treatment with Ca ionophore A23187 and then incubated with IVMD101 containing cycloheximide. Single donor cells (fibroblasts derived from an adult cow) were placed into the perivitelline space of the enucleated oocytes and the two cells were fused by electrofusion. The nuclear transfer embryos were cultured in IVD101 medium without bovine cumulus/granulosa cell co-culture in a humidified atomosphere of 5% CO2/95% air at 38.5�C. Oxygen consumption rates by whole embryos were quantified individually by the SECM measuring system. Some of these embryos were prepared for observation by transmission electron microscopy. Statistical analyses were performed using one-way analysis of variance (ANOVA) and Fisher
220 CpG METHYLATION PROFILE OF ADIPOGENIC AND ENDOTHELIAL GENE PROMOTERS IN HUMAN ADIPOSE STEM CELLS SUGGESTS A RESTRICTIVE ENDOTHELIAL DIFFERENTIATION POTENTIAL
Authors: Boquest A. C. , Noer A. , Sørensen A. L. , Vekterud K. , Collas P.
Published: December 2006

Mesenchymal stem cells (MSCs) have received intense research interest due to their perceived potential application in regenerative medicine; nevertheless, MSCs are primarily restricted to form mesodermal cell types. Adipose stem cells (ASCs) with a CD34+ CD105+ CD45– CD31– immunophenotype can be obtained in an uncultured state with high purity from the stromal vascular fraction of human liposuction material (Boquest et al. 2005 Mol. Biol. Cell 16, 1131–1141). While ASCs differentiate readily into adipocytes, their endothelial lineage commitment has been scarcely reported, and controversy remains regarding ASC contribution to vascularization. To address the epigenetic commitment of ASCs to adipogenic and endothelial lineages, we carried out a bisulfite sequencing analysis of CpG methylation in the promoters of adipogenic (LEP, PPARG2, FABP4, LPL), endothelial (CD31, CD144), and myogenic (MYOG) genes in freshly isolated and in clonal ASC cultures in relation to gene expression and differentiation potential. Uncultured ASCs display mosaic hypomethylation of adipogenic promoters, in contrast to MYOG, CD31, or CD144 which are methylated (Noer et al. 2006 Mol. Biol. Cell 17, in press). Nevertheless, CpG methylation does not reflect transcriptional status of these genes in undifferentiated cells. Culture and adipogenic differentiation of ASCs maintains the hypomethylated profile of adipogenic promoters and the hypermethylation of non-adipogenic promoters. Endothelial stimulation of ASCs in methylcellulose elicits tubule-like networks, up-regulation of CD31 and CD144, and restrictive induction of a CD31+ CD144+ immunophenotype. Discrete and lineage-specific changes in CpG methylation in the CD31 and CD144 promoters take place but no global demethylation that marks endothelial cells occurs. Promoters not involved in endothelial differentiation retain a methylation profile characteristic of undifferentiated cells. Hypermethylation of CD31 and CD144 suggests a restricted commitment of ASCs to the endothelial lineage. This contrasts with hypomethylation of adipogenic promoters which reflects a propensity toward adipogenic differentiation. Despite the up-regulation of lineage-specific transcripts, overall maintenance of promoter methylation after adipogenic, osteogenic, and endothelial differentiation suggests the maintenance of an epigenetic signature characteristic of undifferentiated cells. Analysis of CpG methylation at lineage-specific promoters should provide a robust assessment of epigenetic commitment of stem cells to a specific lineage.
222 LOSS OF IMPRINTS OF PARTHENOGENETIC EMBRYONIC STEM CELLS IN MURINE CHIMERAS
Authors: Horii T. , Kimura M. , Morita S. , Nagao Y. , Hatada I.
Published: December 2006

Mammalian parthenotes with the 2 maternal genomes cannot develop to term. By contrast, chimeras produced by parthenogenetic and normal embryos can develop to term. However, parthenogenetic cells contribute to restricted cells and body weights of the chimeras are reduced. These effects are due to aberrant expressions of imprinted genes, with complete methylation of the maternally methylated genes and complete loss of the paternally methylated genes. On the other hand, parthenogenetic ES (PGES) chimeras show more normal tissue contribution of donor cells and body weight compared to parthenogenetic embryo (PG) chimeras. To elucidate the epigenetic mechanisms underlying this, we analyzed the epigenetic status of maternally methylated genes in murine PG and PGES chimeras. To make parthenogenetic chimeras, PG and PGES cells which express green fluorescent protein (GFP) were introduced into normal host embryos. Mouse embryonic fibroblasts (MEFs) from E13.5 chimeric fetuses were sorted by the fluorescence-activated cell sorter (FACS). Methylation status of parthenogenetic cells was analyzed by combined bisulfite restriction analysis (COBRA) and bisulfite sequencing. Methylation of maternally methylated genes, Peg1/Mest, Snrpn, and Igf 2r, was almost totally maintained in PG chimeras. Average methylatation percentages of PG-derived MEFs were 80% in Peg1/Mest, 84% in Snrpn, and 81% in Igf 2r (n = 6). In contrast, methylation in some PGES chimeras was partially reduced to normal level in all 3 genes (10–45%, n = 7). To clarify whether demethylation is correlated with expression of the imprinted genes, gene expression was analyzed by quantitative real-time RT-PCR. Among maternally imprinted genes, Peg1/Mest and Snrpn are expressed from the paternal allele, whereas Igf 2r is expressed from the maternal allele. Therefore, in parthenogenetic cells, loss of imprints is expected to up-regulate Peg1/Mest and Snrpn expression, and down-regulate Igf 2r expression. In fact, PGES-derived MEFs were up-regulated in Peg1/Mest and Snrpn expression, and down-regulated in Igf 2r expression. This study revealed that variations of imprint status were observed frequently in somatic cells of PGES cell origin. Demethylation could have occurred during establishment and/or maintenance of PGES cells. This demethylation that occurred in PGES cells could reprogram the maternally methylated imprinted genes and improve tissue contribution and body weight to normal level. The PGES cells with reprogramming ability might be utilized for fertility treatment and regenerative medicine.
223 COMPARISON OF NEURAL DIFFERENTIATION BETWEEN RAT AND CAT MESENCHYMAL STEM CELLS UNDER CHALLENGE BY THE SAME NEUROINDUCTION AGENT
Authors: Jin G. Z. , Yu X. F. , Cho S. J. , Park G. Y. , Lee Y. J. , Yin X. J. , Kong I. K.
Published: December 2006

Bone marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate into a variety of cell types, including osteocytes, chondrocytes, and adipocytes. Moreover, MSCs have the capacity to differentiate into neural lineages. The present study aimed to investigate the differences between the expression profiles during neural differentiation of rat and cat MSCs. MSCs in the 4th passage, which were isolated from the femurs and tibias by standard methodology, were used in our study. Culture media was divided into pre-induction medium, which consisted of DMEM-HG, 10% fetal bovine serum, 10 ng mL-1 of basic fibroblast growth factor (bFGF), and 1 mM �-mercaptoethanol, for 24 h, and neuron induction medium, which was composed of DMEM-HG, 2% DMSO, 200 �M BHA, 25 mMKCl, 10 �M forskolin, 5 �g mL-1 insulin-transferrin-sodium selenite, and 20 ng mL-1 bFGF, for 24 h. Thereafter, cell morphology and growth traits were determined by light microscopy imaging and by examining the cell-surface antigen profile and differentiation repertoire by immunocytochemistry, respectively. Regarding the expression of 3 MSC-related surface antigens, rat cells were positive for CD18 and CD44, whereas cat cells expressed CD9 and CD44. Under proneurogenic conditions, rat neuron-like cells progressively increased at 30 min post-induction, peaked at 1 h, and gradually declined after 12 h. At 24 h after neural induction, there were still some neuron-like cells. Meanwhile, cat cells were expressed increasingly during the first hour, peaked at 2 to 3 h, were sustained for 8 h after neural induction, and then gradually declined.A few neuron-like cells remained until 24 h post-induction. In neural differentiation, rat MSCs were positive for �III-tubulin, NF-L, NF-M, NF-H, trkA, and vimentin; cat MSCs were negative for �III-tubulin and NF-H but positive for NF-L, NF-M, trkA, and vimentin. Both MSCs were negative for oligodendrocyte markers. Our results revealed that there is variation in neural differentiation sensitivity due to species type. Rat cells were more sensitive in response to neuroinduction agents, maintained their morphology for a longer time, and expressed relatively mature neuronal markers. On the contrary, the sensitivity of cat cells was weaker, and survival time was shorter compared to that of the rat cells. The cat cells expressed immature neuronal markers. The present data suggest a significant aspect of the culture of MSCs from higher-grade species.
This work was supported by KOSEF (Grant ? M10525010001-05N2501-00110).
226 CELL LINE-DEPENDENT GENE EXPRESSION PROFILES IN MOUSE EMBRYONIC STEM CELLS
Authors: Mamo S. , Kobolak J. , Becker S. , Horsch M. , Beckers J. , Dinnyes A.
Published: December 2006

Molecular phenotyping studies carried out so far on different embryonic stem cells (ESC) have focused mainly on the identification of molecular markers responsible for pluripotency. Unlike these, the goals of our study were to compare and functionally characterize the gene expression profiles of R1 ESC established from F1 (129X1/SvJ � 129S1) blastocysts (Nagy et al. 1993 PNAS 90, 8424–8428) and HM-1 ESC established from an inbred strain (Selfridge et al. 1992 Somat. Cell Mol. Genet. 18, 325–336), as our earlier study showed performance variations between these cells. ES cells were cultured on a mitomycin C-treated mouse embryonic fibroblast feeder cell layer. Cells were grown in standard ES cell medium changed daily [high glucose DMEM (GIBCO-Invitrogen, Carlsbad, CA, USA) supplemented with Na pyruvate (0.11% w/w; GIBCO-Invitrogen), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, 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 �g mL-1; Sigma-Aldrich)]. Total RNA was isolated from aliquots of R1 ES cells at passage 13 and HM-1 ES cells at passage 23 using RNeasy Midi kit (Qiagen, D�sseldorf, Germany) procedures. Fifteen �g of total RNA each from the contrasting samples were used for reverse transcription and labeling with either Cy3 or Cy5 dyes (Amersham, Buckinghamshire, UK). The labeled samples were dissolved in hybridization buffer, added to the cDNA arrays (custom produced at GSF) containing over 21 000 sequences, and hybridized for 17 h at 42�C. The microarray results of 4 independent hybridizations were analyzed, and differentially regulated genes were identified. Finally, the results of 4 randomly selected genes were verified by real-time PCR analysis. The analysis revealed 55 transcripts that showed significant variation (P < 0.01) between the 2 ES cell lines. Of these, 8 transcripts were up-regulated and the rest down-regulated in the HM-1 ES cells. Most of these genes were over-represented in important biological processes such as growth and development (21%), cell organization and biogenesis (11%), regulatory roles (21%), and organogenesis (14%). Moreover, the verification analysis using real-time PCR has confirmed the results of microarray. Thus, based on the detailed analysis, and confirmation of the results with independent analysis, it is possible to conclude that the expression profile reflected the true molecular variations between the 2 ES cell lines, and the identified transcripts can serve as molecular markers that explain biological differences between the 2 ES cell lines.
This work was supported by EU FP6 (MEXT-CT-2003-509582 and 518240), Wellcome Trust (Grant No.070246), and Hungarian National Science Fund (OTKA T046171).
229 DERIVATION OF PRESUMPTIVE GONOCYTES IN VITRO FROM PRIMATE EMBRYONIC STEM CELLS
Authors: Teramura T. , Kawata N. , Takehara T. , Fujinami N. , Takenoshita M. , Matsumoto K. , Saeki K. , Sagawa N. , Hosoi Y. , Iritani A.
Published: December 2006

Embryonic stem cells (ESCs) of nonhuman primates are important for research into human gametogenesis, because of similarities between the embryos and fetuses of nonhuman primates and those of humans. Recently, the formation of germ cells from mouse ESCs in vitro has been reported. In this study, we established cynomolgus monkey ES (cyES) cell lines and attempted to induce their differentiation into germ cells in order to obtain further information on the development of primate germ cells by observing the transcripts of some markers reported as specific for germ cells. CyES cell lines were established using blastocysts produced by intracytoplasmic sperm injection (ICSI). For inducing superovulation, females were treated with 25 IU kg-1 pregnant mare serum gonadotropin once a day for 9 days, followed by 400 IU kg-1 hCG. Oocytes were collected at 40 h after injection of hCG. After sperm injection, embryos were cultured in mCMRL medium to the blastocyst stage. For cyES cell establishment, inner cell masses (ICMs) were isolated by immunosurgery. The ESC colonies developed at about 10 days after ICM plating, and 3 cell lines were successfully established (3/11; 27.3%). All cell lines expressed Oct3/4, SSEA-4, and ALP activity. These ESCs formed teratomas containing 3 different embryonic layers when injected into SCID mice. And the cells could be passaged over 50 times without losing their original properties. To observe in vitro gametogenesis, we attempted to induce differentiation by non-adherent conditions. When cyES cells differentiated spontaneously, the aggregated structures (i.e. embryoid bodies; EBs) accumulated vasa, the expression of which is restricted to germ cells, and some meiotic markers such as dmc1 and sycp1 that exist only in synaptonemal complexes in meiosis. The existence of these markers was also confirmed by immunocytochemistry on cryosections. Interestingly, these products expressed oct4 and nanog again at Day 16, though the expression of both genes diminished at once with onset of differentiation. In vivo, it is reported that vasa, oct4, and nanog are expressed in migrating PGCs, posibly throughout the development of germ cells into spermatocytes/oocytes. Given the results obtained with the meiotic markers, it is possible that developing germ cells such as PGCs or gonocytes could be formed in cynomolgus EBs as in previous cases with mouse or human EBs. These results demonstrate that cyES cells might contribute to putative germ cells in vitro by differentiating into EBs and could be used as a model for studying mechanisms of germ cell development.
This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.
231 HISTOCOMPATIBLE PARTHENOGENETIC EMBRYONIC STEM CELLS
Authors: Yabuuchi A. , Kitai K. , Takeuchi A. , Lerou P. , Ng K. , West J. , Daley G.
Published: December 2006

Organ or tissue transplantation is the preferred treatment for numerous diseases but is hindered by immunologic barriers. Genetically matched pluripotent embryonic stem cells generated via nuclear transfer (ntES cells) or parthenogenesis (pES cells) are possible sources of histocompatible cells and tissues. We have developed two ways of isolating pES cells that carry the full complement of major histocompatibility complex (MHC) antigens of the oocyte donors. One method entails activation of oocytes after blockade of karyokinesis in meiosis II, followed by selection of predominantly homozygous pES cells that have undergone recombination in their MHC antigen region to restore the heterozygous maternal MHC genotype (parthenote recombinant, or prES cells). The second method involves activation of immature oocytes after blockade of karyokinesis of meiosis I, followed by selection of predominantly heterozygous pES lines that retain the MHC genotype of the oocyte donor (parthenote clone recombinant, or pcrES cells). The cells are pluripotent by several criteria: teratoma formation, in vitro differentiation into hematopoietic elements, and high-level skin chimerism in blastocyst chimeras. Breeding of 8 founder females and examination of over 700 progeny failed to demonstrate germ line transmission of the pES cells. Injection of over 50 tetraploid embryos with these lines and embryo transfer have failed to support full gestational development. However, differentiated tissues from these pluripotent ES cells engraft when transplanted into genetically matched immunocompetent recipients, demonstrating that selected pES cells can serve as a source of histocompatible tissues for transplantation.
232 SERUM-FREE DERIVATION OF CAT EMBRYONIC STEM-LIKE CELLS DERIVED FROM IN VIVO-PRODUCED BLASTOCYSTS ON CAT EMBRYONIC FIBROBLAST FEEDERS
Authors: Yu X. F. , Jin X. J. , Choi D. S. , Jung D. K. , Choi B. H. , Cho S. G. , Kong I. K.
Published: December 2006

Fetal bovine serum (FBS) has usually been used as a component of the medium for the culture of embryonic stem (ES) cells. However, FBS contains undefined factors, which promote cell proliferation and occasionally stimulate differentiation of ES cells. To establish an effective culture system for the derivation and maintenance of cat ES cells, we compared the effects of KnockoutTM Serum Replacement (Invitrogen, Seoul, South Korea)-supplemented medium (KSR-medium) and FBS-supplemented medium (FBS-medium) on the attachment and formation of primary ES-like cell colonies of isolated inner cell mass (ICM) derived from in vivo-produced blastocysts on mitotically inactivated cat embryonic fibroblast (CEF) feeders. An inseminated domestic female cat underwent ovariohysterectomy at Day 7 post-insemination, after which blastocysts were flushed from the uterine horns. Inner cell mass (ICM) was mechanically isolated from the in vivo-matured blastocysts (n = 22) and cultured in KSR-medium or FBS-medium on a CEF feeder layer at 39�C, 5% CO2 in air. KSR-medium or FBS-medium consisted of knockoutTM DMEM supplemented with 20% KSR or FBS, 100 IU mL-1 penicillin/streptomycin, 2 mM l-glutamine, 1� MEM non-essential amino acids, 100 �M �-mercaptoethanol, and 1000 IU mL-1 recombinant mouse LIF. The results indicate that incidence of primary ICM attachment (91.6 � 14.4 vs. 55.5 � 9.6% in KSR- and FBS-medium, respectively; P < 0.05) and ES-like cell colony formation (63.8 � 12.7 vs. 27.7 � 4.8% in KSR- and FBS-medium, respectively; P < 0.05) is significantly affected by the ES cell culture medium. However, cat ES-like cells in KSR-medium did not actively proliferate in the primary culture. The number of cat ES-like cells in a colony after 5 days of culture was significantly higher in FBS-medium than in KSR-medium (749 � 82 vs. 383 � 55% in FBS- and KSR-medium, respectively; P < 0.05). A total of 10 ES-like cell colonies were formed in KSR-medium (n = 7) and FBS-medium (n = 3), and these expressed high levels of alkaline phosphatase activity (AP). Immunostaining analysis was positive for the ES cell-markers, Oct-4 and SSEA-3. To our knowledge, this is the first reported isolation of cat ES-like cells on CEF feeders in the absence of serum. A culture system of embryo-derived stem cells using serum-free medium may make it possible to directly examine the effects of factors added to culture medium on isolation and maintenance of cat ES cell lines in vitro.
This work was supported by KOSEF (grant # M105250100 01-05N2501-00110).
234 QUANTITATION OF BOVINE VIRAL DIARRHEA VIRUS ASSOCIATED WITH SINGLE ZONA PELLUCIDA-INTACT IN VIVO-DERIVED BOVINE EMBRYOS
Authors: Gard J. A. , Givens M. D. , Galik P. K. , Stringfellow D. A. , Riddell K. P. , Marley S. E. , Whitlock B. K.
Published: December 2006

Quantitation of bovine viral diarrhea virus (BVDV) associated with individual transferable embryos is prerequisite to a thorough assessment of the risk for transmission of BVDV via embryo transfer. One objective of this study was to determine the proportion of in vivo-derived bovine embryos that remained virus-positive after artificial exposure to a high-affinity strain of BVDV and thorough washing. A second objective was to determine the quantity of virus associated with these individual embryos. A total of 87 zona pellucida-intact, Day 7, in vivo-derived bovine embryos were exposed to a type 1 noncytopathic strain of BVDV (SD-1) and washed according to International Embryo Transfer Society recommendations. Subsequently, individual embryos were sonicated, and the RNA was extracted from the sonicate fluids and stored at -80�C until assayed using a real-time quantitative polymerase chain reaction (QPCR). Twenty-six percent (23/87) of the embryos contained virus. The average quantity of virus associated with individual embryos after viral exposure and washing was 1.12 viral copies per 5 �L (SD = 1.57 copies 5 per �L-1; SEM = 0.33 copies 5 per �L-1). Assessment of data using tolerance intervals (P = 0.05) indicates that 90% of contaminated embryos will be associated with ≤4.64 viral copies per 5 �L, whereas 99% of contaminated embryos will be associated with ≤6.62 copies per 5 �L. Obviously, only extremely small quantities of virus were associated with less than one-third of the embryos tested. Based on previous research, it is presumed that this virus is associated with the outer layers of the zona pellucida. A logical next step in the risk assessment would be to determine if these quantities of zona-associated virus are sufficient to infect na�ve recipients and/or embryonic cells after embryos are transferred. Further, similar efforts should be made to estimate the quantity of virus associated with in vitro-derived, zona pellucida-intact, bovine embryos after exposure to the same high-affinity strain of virus and washing.
236 NORMAL REPRODUCTIVE CAPACITY OF HEIFERS RESULTING FROM TRANSFER OF IN VITRO-FERTILIZED EMBRYOS CULTURED WITH AN ANTIVIRAL COMPOUND
Authors: Givens M. D. , Stringfellow D. A. , Riddell K. P. , Galik P. K.
Published: December 2006

Bovine viral diarrhea virus (BVDV) replicates in embryo culture systems and remains associated with developing IVF bovine embryos despite washing and trypsin treatment. Previous research (Givens et al. 2006 Theriogenology 65, 344–355) determined that 2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan (DB606) can be added to an in vitro embryo production system to prevent replication of BVDV without inhibiting embryonic development. The objective of this research was to assess sexual development and future reproductive capacity of heifers resulting from embryos that had been exposed to DB606 while developing in vitro. Presumptive zygotes that resulted from in vitro fertilization of oocytes were cultured for 7 days in medium supplemented with 0.4 �M DB606 or medium lacking the antiviral agent. All blastocysts were individually transferred nonsurgically into the uterus of a synchronized recipient. Additional control pregnancies were established by natural breeding. At 20 to 27 months of age, 7 heifers resulting from each treatment were simultaneously exposed to a fertile bull in a single pasture during a 63-day breeding season. All but 2 heifers subjected to natural breeding were pregnant 28 days after removal of the bull. One pregnancy was aborted between 5 and 7 months of gestation from a heifer that had undergone IVF without exposure to DB606. All offspring were normal upon clinical observation at birth. One heifer resulting from IVF with exposure to DB606 would not allow its calf to nurse adequate quantities of colostrum; thus, this calf was removed from the study. Adjusted 205-day weaning weights for offspring were evaluated using ANOVA and Tukey Kramer test for pair-wise comparisons. Offspring of dams treated or untreated with DB606 during IVC or produced by natural breeding exhibited mean adjusted 205-day weaning weights of 194, 181, and 170 kg, respectively (not significantly different). Results of this research using a limited number of animals demonstrates that exposure to DB606 during the first 7 days after fertilization does not appear to cause delayed toxic effects that would impair the future reproductive capacity of heifers.
238 EFFICACY OF RECOMBINANT TRYPSIN AGAINST BOVINE HERPESVIRUS-1 ASSOCIATED WITH IN VITRO-DERIVED PORCINE EMBRYOS
Authors: Marley M. S. D. , Givens M. D. , Galik P. K. , Riddell K. P. , Stringfellow D. A.
Published: December 2006

TrypLETM (Invitrogen, Carlsbad, CA, USA) is a recombinant, fungal, trypsin-like protease that is used as a substitute for porcine-origin trypsin in cell culture procedures. It is stable at room temperature and does not present the same risk of contamination as animal-origin trypsin. Previously, TrypLE SelectTM (10X) was shown to remove bovine herpesvirus-1 (BHV-1) from Day 7 in vivo-derived embryos (Marley et al. 2006 Reprod. Fert. Dev. 18, 213–214). The objective of this study was to determine if the same treatment would effectively remove BHV-1 from Day 7 zona pellucida-intact, in vitro-derived porcine embryos. Day 7 in vitro-derived morulae and blastocysts and non-fertile or degenerate embryos (NFD) were washed according to the International Embryo Transfer Society protocol. One group of 10 NFD was not exposed to virus and served as the negative control. The remaining embryos and 10 NFD were exposed to 106–108 PFU/mL BHV-1 (Colorado strain) for 1 h. Following exposure, one group of 10 NFD was washed and served as the positive control. The remaining developed embryos were divided into groups of 10 and washed and treated as described in Table 1. Following treatment, the embryos were sonicated in groups of 5 and assayed by virus isolation. The negative control embryos, as well as the embryos treated with porcine-origin trypsin, TrypLE Select (10X) for 7 min, and TrypLE Select (10X) diluted 1 : 2 for 10 min, were negative for virus. The positive control embryos in addition to the other treatments were positive on virus isolation (Table 1). Although, TrypLE Select (10X) does have some antiviral effect when used for 10 min, it was not completely effective, as shown by the positive virus isolation results of one group of 10 embryos. The groups treated with TrypLE Select (10X) diluted 1 : 2 for 10 min were negative for virus; however, if a larger sample size had been tested, positive groups might have occurred. Though using a recombinant trypsin product would be beneficial over using an animal-origin product, it is not known if TrypLE Select (10X) would render a single IVF embryo free of infectious virus. Further research would also need to be performed to assess the viability of embryos following treatment with TrypLE Select (10X). In addition, other recombinant trypsin products need to be evaluated to determine their efficacy against BHV-1 associated with IVF embryos.

Table 1.Effect of recombinant trypsin-like proteases on BHV-1 virus in porcine embryos
240 RECOMBINANT BOVINE TRYPSIN MADE IN MAIZE INACTIVATES BOVINE HERPES VIRUS-1 ADSORBED TO THE BOVINE ZONA PELLUCIDA
Authors: Seidel, Jr G. E. , Turk M. L. , Gordy P. W. , Bowen R. A.
Published: December 2006

For embryo culture, it is often desirable to use media that are devoid of animal products. One problem is that the trypsin required for treatment of embryos for export is an animal product. Therefore, we studied the efficacy of recombinant trypsin produced from the bovine gene in maize (TrypzeanTM, Sigma-Aldrich Corp., St Louis, MO, USA) for inactivating bovine herpes virus-1 (BHV-1), initially without ova present. We found that 525 U mL−1 Trypzean is equivalent in efficacy to the standard 0.25% bovine trypsin treatment. Active virus was titrated on MDBK cells using a standard plaque assay. Next, ova recovered from superovulated cows were incubated for 45 min with BHV-1-infected cells for virus adsorption to the zona pellucida. Both unfertilized ova (n = 26) and embryos (n = 22) were allotted to 4 treatments: (1) control, not exposed to virus; (2) exposed to virus and then washed 10×; (3) treatment 2 plus trypsin treatment as recommended in the IETS Manual; and (4) treatment 2 plus 525 U Trypzean mL−1 and 1 mM EDTA substituted for the 4th and 5th washes of 1 min each; the 6th wash of 10 washes was with soybean trypsin inhibitor at 80 µg mL−1 (Sigma). The chemically defined medium used for handling ova was Syngro® (AB Technology, Pullman, WA, USA), except that trypsin and inhibitor were made up in Hank's balanced salt solution with 0.2% polyvinyl alcohol, but without Ca2+ or Mg2+. Ova were stored in 0.5 mL at −80°C until sonication and inoculation onto MDBK cells in duplicate undiluted and at 0.1× dilution. The virus was allowed to adsorb for 45 min, and then overlaid with MEM containing 0.5% agarose, 5% FBS, and antibiotics. Two days later an identical overlay was added, but containing 0.005% neutral red; plaques were counted the following day. Unfertilized ova and embryos led to similar results, which are pooled. No virus was detected in the 5 control ova not exposed to virus. As has been shown by others, BHV-1 remained adhered to zonae even after 10 washes (Table 1). In contrast, treatment with Trypzean or the IETS trypsin protocols reduced this to near zero. We thus confirm that trypsin treatment is effective in inactivating BHV-1 adhered to bovine zonae pellucidae, whether trypsin is derived from animals or genetically engineered plants. We have found that Trypzean is more efficacious than some ‘trypsin-like’ enzymes for this purpose.

Table 1.Mean plaque-forming units (pfu) per ovum incubated with virus (2 replicates per ovum pooled)
241 TIME POINTS FOR IN VITRO MATURATION OF LION (PANTHERA LEO) OOCYTES
Authors: Adamiak S. , Bartels P.
Published: December 2006

The objective of the study was to establish a time point for successful in vitro maturation of lion (Panthera leo) oocytes using a model developed for the domestic cat (Gomez et al. 2003 Theriogenology 60, 239–251). As part of a game reserve management program, one adult free-ranging lioness (6–7 years old) and her 3 sub-adult cubs (18 months old) were chemically immobilized with 500 mg of a combination of tiletamine and zolazepam (Zolatil 100�; Virbac SA, Carras, France) by remote injection (Dan-inject�; Dan-Inject ApS, Borkop, Denmark) and within 20 min euthanized using 8 g sodium pentobarbitone (Euthapent� KruVet, SA). Ovaries collected from the adult and 2 sub-adult females were transported to a laboratory in a flask containing warm (37�C) sterile saline. Within 2 h of collection, all visible follicles were aspirated using a 21G needle attached to a 5-mL syringe. To increase the number of recovered oocytes, ovaries were minced using a scalpel blade in a 60-mm Petri dish containing warm search medium (HEPES-buffered TCM-199, 2.2 mM Ca lactate, 0.36 mM pyruvate, 2 mM glutamine, 1.12 mM cysteine, 0.3% w/v fatty acid-free BSA, and 50 �g mL-1 gentamicin). A total of 33 and 54 oocytes were recovered from the adult and sub-adult females, respectively, and cultured in 35-mm Petri dishes containing 3-mL of maturation medium (sodium bicarbonate-buffered TCM-199, 1 IU mL-1 hCG, 0.5 IU mL-1 eCG, 2.2 mM Ca lactate, 0.36 mM pyruvate, 2 mM glutamine, 1.12 mM cysteine, 0.3% w/v fatty acid-free BSA, and 50 �g mL-1 gentamicin). Petri dishes containing oocytes were enclosed in a sealed plastic bags, filled with a humidified gas mixture of 5% CO2, 5% O2, and 90% N2, and incubated at 38.8�C. After 26, 32, or 38 h of incubation, groups of 29 oocytes were fixed in 3 : 1 ethanol : acetic acid solution and stored at 4�C for 48 h. Fixed oocytes were stained with 1% w/v orcein and visualized with phase-contrast microscopy. Oocytes in telophase I or metaphase II were classified as mature. Each ovary had an average of 22.5 � 3.0 antral follicles, where 8.8 � 2.0 were 2-3 mm, and 13.8 � 1.5 were 1 mm in diameter. There were no CLs present. Out of 87 oocytes recovered, 24.0 � 3.7% had a uniform cytoplasm and >3-4 layers of cumulus cells, 42.2 � 6.0% had a uniform cytoplasm and 2 or less layers of cumulus cells, and 33.8 � 9.7% had no cumulus cells attached. None of the oocytes were mature at 26 h, but at 32 h and 38 h, the percentage of matured oocytes significantly (P < 0.05) increased to 63.9 � 13.9% and 80.4 � 7.1%, respectively. These results indicate that the domestic cat system used herein can be successfully applied for in vitro maturation of lion oocytes. However, unlike oocytes from a domestic cat, lion oocytes required a culture period of 32 to 38 h to reach metaphase II. Further studies are required to confirm these findings and to test fertilization rates of such matured oocytes, and their ability for further development.
243 IN VITRO PRODUCTION OF LLAMA EMBRYOS BY IVF OR ICSI
Authors: Conde P. A. , Herrera C. , Chaves M. G. , Giuliano S. M. , Director A. , Trasorras V. L. , Pinto M. , Sarchi M. I. , Stivale D. , Rutter B. , Agüero A. , Miragaya M. H. , Pasqualini R. S.
Published: December 2006

Interest in South American camelids has increased in the last few years. Assisted reproduction techniques, such as in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) with epididymal spermatozoa, have shown poor results in these species (Tibary et al. 2005 Theriogenology 64, 618–638). The aim of the present study was to compare the efficacy of IVF vs. ICSI for in vitro embryo production in llama (Lama glama) using oocytes collected from ovarian follicles of different sizes. A total of 193 oocytes were collected from 223 follicles aspirated from 21 adult females by flank laparotomy after ovarian stimulation. Before aspiration, the diameter of each follicle was measured, and oocytes from each female were randomly assigned to either IVF or ICSI treatment. Semen samples were collected by electroejaculation and incubated in 25% (v/v) collagenase solution at 37°C to reduce viscosity. For IVF, spermatozoa were either non-treated or treated with heparin, penicillamine, and hypotaurine as capacitating agents. For ICSI, some oocytes were activated immediately after sperm injection with 5 µM of ionomycin for 10 min and 2 mM of 6-DMAP for 3 h, while others were subjected only to sperm injection. Spermatozoa used for ICSI were not treated with capacitating agents. All presumptive zygotes were cultured in SOFaas for 8 days. The percentage of total oocytes and mature (MII) oocytes recovered from follicles <7 mm and >7 mm in diameter were compared in each female. The proportion of oocytes inseminated via IVF and ICSI that cleaved and developed to the blastocyst stage was compared. The proportion of total oocytes, MII-oocytes, cleaved embryos, and blastocysts were compared between treatments by chi-square and Fisher's tests. The percentages of total (77/100; 77%) and MII (9/31; 29%) oocytes collected from <7 mm follicles were significantly lower than those of total (116/126; 92%) and MII (43/55; 78%) oocytes collected from >7 mm follicles (P < 0.01). The highest cleavage rates were observed in oocytes collected from follicles >7 mm in diameter and fertilized by IVF with (56%) or without (50%) capacitating agents; these rates were significantly different from those of the other treatments (P < 0.05, Table 1). Further studies will determine if the present results can be obtained with a higher number of oocytes. The results of the present study provide the first demonstration that Lama glama embryos can be produced in vitro using fresh semen. In addition, we have provided the initial description of blastocyst development after culture of ICSI-derived embryos in a defined medium.

Table 1.Cleavage and blastocyst formation in different-sized llama oocytes inseminated via IVF or ICSI