bluuebunny/crossref_metadata_2025 huggingface dataset:
56 TRANSGENESIS BY HANDMADE CLONING USING EGFP-TRANSFECTED YUCATAN FIBROBLASTS
Authors: Kragh P. M. , Du Y. , Li J. , Zhang Y. , Bolund L. , Vajta G.
Published: December 2006

Somatic cell nuclear transfer (SCNT) offers the possibility of pig transgenesis. Importantly, genetic manipulations can be performed in cells isolated from special breeds followed by SCNT into enucleated oocytes isolated from slaughterhouse ovaries. In the present study, we established production of Yucatan blastocysts by the handmade cloning (HMC) technique using non-transgenic fibroblasts from the Yucatan miniature pig, and produced transgenic blastocysts using enhanced green fluorescent protein (EGFP)-positive Yucatan fetal fibroblasts. For transgenesis, Yucatan fibroblasts from a 40-day old fetus were transfected with a vector containing an EGFP gene and a neomycin-resistance selection gene by lipofection. Well separated neomycin-resistant colonies were isolated, expanded, and used for HMC. For HMC, cumulus–oocyte complexes were aspirated from ovaries of slaughterhouse sows and matured for 41 h. Subsequently, the cumulus cells were removed in hyaluronidase, and zonae pellucidae were partially digested by incubation in pronase. Oocytes with a visible polar body (PB) were subjected to oriented bisection. Less than half of the cytoplasm adjacent to the PB was removed with a microblade. The remaining parts, i.e. cytoplasts, were used as recipients for embryo reconstruction. Reconstructed embryos were produced by a two-step fusion procedure. At the first step, one cytoplast was fused with one fibroblast in the absence of Ca2+. After one h, the cytoplast-fibroblast pair and another cytoplast were fused and activated simultaneously in the presence of Ca2+, and subsequently cultured in cytochalasin B and cycloheximide for 4 h. The development of reconstructed embryos to the blastocyst stage was determined after 7 days of in vitro culture. When using non-transgenic and EGFP-positive Yucatan fetal fibroblasts, the rate of blastocyst formation (mean � SEM) were 36 � 7% (36/102) and 42 � 7% (32/77), respectively. In conclusion, the HMC technique was very efficient for production of blastocysts of Yucatan miniature pig origin using both non-transgenic and EGFP-positive fetal fibroblasts.
59 HIGHER BLASTOCYST FORMATION OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DOES NOT GUARANTEE BETTER PREGNANCY IN PIG
Authors: Lee E. , Song K. , Jeong Y. , Hyun S.
Published: December 2006

Generally, blastocyst (BL) formation and embryo cell number are used as main parameters to evaluate the viability and quality of in vitro-produced somatic cell nuclear transfer (SCNT) embryos. We investigated whether in vitro development of SCNT pig embryos correlates with in vivo viability after transfer to surrogates. For SCNT, cumulus–oocyte complexes (COCs) were matured in TCM-199 supplemented with follicular fluid, hormones, EGF, cysteine, and insulin for the first 22 h and in a hormone-free medium for 18 h. Three sources of pig skin cells were used as nuclear donor: (1) skin fibroblasts of a cloned piglet that were produced by SCNT of fetal fibroblasts from a Landrace × Yorkshire × Duroc F1 hybrid (LYD), (2) skin fibroblasts of a miniature pig having the human decay accelerating factor gene (hDAF-MP), and (3) skin fibroblasts of a miniature pig with a different strain (MP). MII oocytes were enucleated, subjected to nuclear transfer from a donor cell, electrically fused, and activated 1 h after fusion. SCNT embryos were cultured in a modified NCSU-23 (Park Y et al. 2005 Zygote 13, 269–275) for 6 days or surgically transferred (110–150 fused embryos) into the oviduct of a surrogate that showed standing estrus on the same day as SCNT. Embryos were examined for cleavage and BL formation on Days 2 and 6, respectively (Day 0 = the day of SCNT). BLs were examined for their cell number after staining with Hoechst 33342. Pregnancy was diagnosed by ultrasound 30 and 60 days after embryo transfer. Embryo cleavage was not affected by donor cells (82, 81, and 72% for LYD, hDAF-MP, and MP, respectively), but BL formation was higher (P < 0.05) in hDAF-MP (16%) than in LYD (9%) and MP (6%). MP showed higher (P < 0.05) BL cell number (46 cells/BL) than hDAF-MP (34 cells) but did not show a difference from LYD (37 cells). LYD and MP showed higher pregnancy rates (Table 1) on Days 30 and 60, even though they showed lower BL formation in vitro. Due to a relatively small number of embryo transfers through a limited period, we could not exclude any possible effects by seasonal or operational differences. These results indicated that pregnancy did not correlate with in vitro BL formation of SCNT pig embryos but rather were affected by the source of donor cells.

Table 1.In vivo development of somatic cell nuclear transfer pig embryos derived from different sources of donor cells

This work was supported by the Research Project on the Production of Bio-organs (No. 200506020601), Ministry of Agriculture and Forestry, Republic of Korea.
61 EFFICIENCY OF TWO ENUCLEATION METHODS FOR THE PRODUCTION OF TRANSGENIC PIG EMBRYOS BY HANDMADE CLONING
Authors: Li J. , Zhang Y.-H. , Du Y.-T. , Kragh P. M. , Purup S. , Pederson A. M. , Villemoes K. , Jørgensen A. L. , Bolund L. , Xue Q.-Z. , Yang H.-M. , Vajta G.
Published: December 2006

Since the successful production of transgenic pigs by somatic nuclear transfer (Lai et al. 2002 Science 295, 1089–1092), more efficient reproduction technologies for transgenic pigs have been in demand. The purpose of our work was to develop an efficient method for production of transgenic embryos by handmade cloning (HMC; Vajta et al. 2001 Cloning 3, 89–95) connected to oriented enucleation to eliminate potential harm of staining and UV illumination at cytoplast selection. After 41–42 h of in vitro maturation, oocytes were further cultured with or without 0.4 µg mL−1 demecolcine for 45 min (i.e. chemically assisted handmade enucleation (CAHE) vs. oriented handmade enucleation (OHE)). Subsequently, the cumulus cells were removed and zonae pellucidae were partially digested. Oocytes with visible extrusion cones or polar bodies attached to the surface were subjected to oriented bisection. The putative cytoplasts without extrusion cones or polar bodies, containing the major part of cytoplasm, were selected as the recipients. Two cytoplasts were electro-fused with one transgenic fibroblast expressing either amyloid precursor protein (APP) or green fluorescent protein (GFP), while non-transgenic fibroblasts were used as control nuclear donors. After activation (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Cloning Stem Cells 7, 199–205), reconstructed embryos were cultured in porcine zygote medium-3 for 7 days. The rates of cleavage and blastocyst cell numbers were recorded on Day 2 and Day 7, respectively. In 5 replicates, the correct bisection efficiency achieved with CAHE was higher compared to that with the OHE method (93 ± 1% vs. 82 ± 2%, respectively; P < 0.05). Table 1 shows that blastocyst rates with APP and GFP transgenic fibroblasts as nuclear donors after CAHE were lower (P < 0.05) compared to those with the OHE method; in contrast, cleavage rates of embryos from different fibroblast donors were similar and so were blastocyst rates of non-transgenic donors after either CAHE or OHE. Our results show that embryos reconstructed from APP and GFP transgenic donors have compromised in vitro developmental rates after CAHE rather than after the OHE method; however, a high efficiency with both enucleation methods was observed when using non-transgenic somatic cells.

Table 1.Comparison of two enucleation methods for the production of transgenic pig embryos
62 EXPRESSION AND DISTRIBUTION OF Oct-4 IN INTERSPECIES-CLONED LONG-TAILED MONKEY (MACACA FASCICULARIS) EMBRYOS
Authors: Lorthongpanich C. , Srirattana K. , Imsoonthornruksa S. , Sripunya N. , Laowtammathron C. , Kumpong O. , Ketudat-Cairns M. , Parnpai R.
Published: December 2006

The transcription factor Oct-4 is one of the important factors for early embryonic development and differentiation in several animal species such as monkey (Mitalipov et al. 2003 Biol. Reprod. 69, 1785–1792) and bovine (Kurosaka et al. 2004 Biol. Reprod. 71, 1578–1582). However, Oct-4 expression and distribution in interspecies somatic cell nuclear transfer (iSCNT) embryo have not yet been studied. Therefore, this study was undertaken to evaluate the Oct-4 distribution in monkey iSCNT embryos at the 2-cell (2C), 4-cell (4C), 8-cell (8C), 16-cell (16C)/morula, hatching (Day 7), and hatched blastocyst stages (Day 8), compared with bovine SCNT embryos. The skin fibroblasts from adult monkey (Macaca fascicularis) and bovine were used as donor cells, and bovine enucleated oocytes were used as recipient cytoplasts. SCNT procedures were performed as previously reported (Laowtammathron et al. 2005 Theriogenology 64, 1185–1196). The result was that monkey iSCNT embryos have cleavage (93.8%; 76/81) and development rates up to the 8-cell stage (76.5%; 62/81) similar to those in bovine SCNT (96.0%; 96/100, and 89.0%; 89/100, respectively). Bovine SCNT developed to morulae (61.5%; 59/96) and blastocysts (41.7%; 40/96) at a higher rate than monkey iSCNT (43.4%; 33/76, and 11.8%; 9/76, respectively). Moreover, bovine SCNT blastocysts showed inner cell mass (ICM) and trophectoderm (TE), but monkey iSCNT embryos formed a blastocoel and ICM and TE could not be clearly seen. Five embryos from each group were used to study Oct-4 expression and distribution by immunocytochemistry. The embryos were incubated with 1 : 1000 anti-mouse Oct-4 polyclonal antibodies (Chemicon International, Inc., Temecula, CA, USA), then rinsed in PBS, and incubated with 1 : 200 FITC-labeled antimouse IgG (Sigma-Aldrich Corp., St Louis, MO, USA), followed by 2 �g mL-1 422-6-Diamidino-2-phenylindole (DAPI) (Sigma); the embryos were then mounted and examined by fluorescence microscopy. The results showed Oct-4 staining in every nucleus of bovine and monkey iSCNT embryos at the 2C, 4C, 8C, and 16C/morula stages but was varied in blastocysts. The Oct-4 staining was reduced in TE cells when half of the embryonic cells hatched out of the zona pellucida (60.0%; 3/5). The Oct-4 staining continued to decrease in the TE of hatched blastocysts (Day 8) but did not affect the ICM. The total cell number of bovine SCNTs at blastocyst stage were 140.9 � 13.2 and, of these, 13.5% (19/140) showed apoptotic nuclei morphologies, whereas the cell numbers of monkey iSCNT early blastocysts had only 15.6 � 2.3 cells and, of these, 19.2% (3/15) showed apoptotic nuclei. It can be concluded from this study that monkey iSCNT embryos show Oct-4 staining in every intact nucleus.
66 SUCCESSFUL ESTABLISHMENT OF PLURIPOTENT ntES CELL LINES FROM AGED MICE
Authors: Mizutani E. , Kishigami S. , Thuan N. V. , Ohta H. , Hikichi T. , Thuy B. H. , Wakayama S. , Sakaide Y. , Wakayama T.
Published: December 2006

Nuclear transfer technique has enabled us to produce cloned animals from somatic cell nuclei in various animal species to date. Moreover, it has been demonstrated that ES cell lines have been established from cloned blastocysts by somatic cell nuclear transfer (ntES cell), irrespective of sex, strains, or organs. These cells are capable of differentiating into all 3 germ layers in vitro, or even into spermatozoa and oocytes in chimeric mice. So ntES cells have gotten a lot of attention recently in the field of regenerative medicine. However, it is unclear whether ntES cells can be established from aged individuals because, in general, the cloning success rate was higher when young donor cells were used, such as fetus cells rather than adult. To answer this question, we tried to establish ntES cell lines from aged mice and then examined their pluripotency. The donor cells were obtained from tail-tip fibroblast cells of 11-month-old to 15-month-old male and female mice. After nuclear transfer, we succeeded in establishing 8 ntES cell lines from 3 aged BDF1 males and 6 ntES cell lines from 2 aged BCF1 females. The normality of these ntES cell lines was examined after passages 5 times. Karyotypes were analyzed using SKY-Fish painting, and pluripotency was examined by chimeric mice formation, in which ntES cells were injected into fertilized ICR blastocysts. As a result, most of the ntES cell lines examined had normal karyotypes, and all of the ntES cell lines tested could contribute to somatic cells of chimeric mice. Now we are examining whether these ntES cells have germ line transmission ability in chimeric mice by natural mating.
69 APPLICATION OF BOVINE AMNIOTIC CELLS FOR PRENATAL GENETIC DIAGNOSIS AND NUCLEAR TRANSFER
Authors: Nagao Y. , Watanabe T. , Furutani R. , Kato Y. , Takahashi R. , Hirabayashi M. , Ueda S. , Kurihara T.
Published: December 2006

Amniotic fluid includes many cells derived from the fetus called 'amniotic cells'. Although these amniotic cells may have much potential as a reproduction or breeding source of the animals, there has been limited study of the potential applications of these cells. We examined the potential of bovine amniotic cells for biotechnological use. Bovine amniotic cells separated from amniotic fluid obtained from a slaughterhouse were prepared for use in all experiments. First, to examine the culture condition of amniotic cells, the cells were cultured in various culture media. Cytologic normality of the cultured cells was analyzed by chromosomal examination and Papanicolaou examination. Second, to examine the potential of cultured amniotic cells for prenatal genetic diagnosis, the cells were used for sexing by PCR. The coincidence of the results with the gender of the fetus from which the cells were derived was examined. Third, we used the cultured cells as donor cells for nuclear transfer, and examined the developmental ability of reconstructed embryos. The normality of the blastocysts derived from the reconstructed embryos was examined by chromosomal examination and transplantation to the recipient heifer. Bovine amniotic cells were cultured successfully in Amnio-max C-100� (GIBCO, Grand Island, NY, USA), which is marketed as culture medium for human amniotic cells. In all cases, the sex of cultured amniotic cells analyzed by PCR was coincident with that of the fetus from which the amniotic cells were derived. The frequencies of cleavage and development to the blastocyst stage of embryos reconstructed from amniotic cells were the same as those of fetal fibroblasts. There were no differences in the normality of chromosomal number between blastocysts derived from amniotic cells and fetal fibroblasts. A blastocyst derived from amniotic cells developed into a fetus after transplantation. DNA microsatellite analysis of the fetus at Day 64 was coincident with that of the fetus from which the amniotic cells were derived. These results indicate that bovine amniotic cells can be successfully cultured in vitro, and the cultured cells precisely reflect the genetic information of the fetus from which the cells were derived. The cultured cells also have developmental ability as donor cells for nuclear transfer. Amniotic cells may have the potential for effective reproduction and breeding using genetic and biotechnological sources.
70 FETAL FIBROBLAST CELLS PERMEABILIZED WITH STREPTOLYSIN O IMPROVE THE RATES OF FUSION AND IN VITRO DEVELOPMENT OF PORCINE RECONSTRUCTED EMBRYOS
Authors: Naruse K. , Shin Y. M. , Quan Y. S. , Park C. S. , Jin D. I.
Published: December 2006

Streptolysin O (SLO) is known to bacterial proteins that form very large pores in the plasma membrane of mammalian cells. SLO has been used in the delivery of proteins into living cells following permeabilization. The objective of this study was to investigate the effect of permeabilization of donor cells using SLO on in vitro development of porcine reconstructed embryos. Porcine fetal fibroblast cells were treated with Ca2+-free DMEM medium containing 200 ng mL−1 of SLO for 50 min before or after trypsinization. Those SLO-treated donor cells were injected into enucleated oocytes, fused with 2 DC pulses (1.2 kV cm−1, 30 µs) and cultured in procine zygote medium-3 (PZM-3) for 6 days. In vitro development of the reconstructed embryos was examined. SLO treatment after trypsinzation significantly increased (P < 0.05) the percentage of fusion rates and blastocyst developmental rates compared with that before trypsinization or in the nontreated group. Additionally there were no significant differences in fusion rates, cleavage rates, blastocyst developmental rates, and total cell number of blastocysts between the SLO-treated group before trypsinzation and the nontreated group. Next, after the trypsinzation treatment, fetal fibroblast cells were incubated in Ca2+-free DMEM containing 200 ng mL−1 of SLO for 0, 30, 50, and 70 min and SLO-treated donor cells were also tested for fusion rate and developmental capability following reconstruction. The 50-min group of SLO-treated cells significantly increased (P < 0.05) the percentage of fusion rates (90.6 vs. 77.6, 85.4, and 78.5%) and blastocyst developmental rates (24.7 vs. 13.5, 11.2, and 13.5%) compared with the other groups (Table 1). However, there was no significant difference in the total cell number of blastocysts among SLO-treated groups. Although cleavage rates the in SLO-treated groups were not significantly different from those of the nontreated group, there the cleavage rates were slightly in SLO-treated groups. In conclusion, permeabilization of porcine fetal fibroblast cells with SLO improves the fusion rates and in vitro development of porcine reconstructed embryos.

Table 1.Effects of SLO treatment of fetal fibroblasts by different exposure times on in vitro development of porcine reconstructed embryos
71 THE DEVELOPMENT OF INTRA- AND INTER-SPECIES CLONED EMBRYOS DERIVED FROM RABBIT OOCYTES: THE EFFECT OF DONOR CELL SOURCES
Authors: Numchaisrika P. , Thongphakdee A. , Rungsiwiwut R. , Pruksananonda K. , Virutamasen P. , Techakumphu M.
Published: December 2006

Generating cloned embryos using rabbit oocytes as the recipient cytoplasm is useful for studying the interaction between a foreign donor nucleus and the recipient cytoplasm. The aims of the study were to (1) investigate the development of inter-species cloned embryos derived from rabbit oocytes, using cow (no. 1 and no. 2), swamp buffalo, pig, and elephant ear fibroblasts as donor cells, and (2) observe the developmental capacity of cloned rabbit embryos derived from adult and fetal fibroblasts. Mature oocytes were collected from superovulated rabbit does by oviductal flushing. A single fibroblast (passages 3–5) in the starvation stage (1–3 days prior to nuclear transfer) was transferred into an enucleated oocyte, which was confirmed by Hoechst staining. Reconstructed oocytes were later fused by electrical stimulation using 3 DC pulses, 3.2 kV.cm-1, for 20 �s. Fused couplets were activated by inducing the same electrical stimulation, and subsequently incubated in activation medium comprised of 5 �g mL-1 cycloheximide, 2 mM mL-1 6-DMAP and 10% fetal bovine serum (FBS), in synthetic oviduct fluid (SOF), for 1 h. They were then cultured in SOF supplemented with 10% FBS for 5 days. Differences in the percentages of fusion and embryo development to a particular stage between source of donor cells were determined by chi-square analysis. The fusion rates of cow no. 1 (62%; 82/133), cow no. 2 (54%; 41/76), buffalo (63%; 53/84), pig (69%; 60/87), elephant (59%; 44/74), adult rabbit (55%; 41/74), and fetal rabbit couplets (70%; 56/80) did not differ significantly. The cleavage rates of cows no. 1 and 2, buffalo, pig, and elephant embryos were 33%, 83%, 57%, 67%, and 100%, respectively. The rabbit fibroblasts derived from an adult gave a significantly lower cleavage rate than those derived from a fetus (54% vs. 95%) (P < 0.05). The developmental rates at morula and blastocyst of cloned embryos reconstructed from fetal rabbit fibroblasts were significantly greater than those reconstructed from adult rabbit, buffalo, pig, and elephant (morula: 39%, 12%, 4%, 7%, and 14%; blastocyst: 23%, 7%, 0%, 3%, and 9%, respectively; P < 0.05). While cow no. 2 donor cells provided a significant embryo development success rate in terms of blastocyst formation, cow no. 1 cloned embryos could not develop beyond the cleavage stage (morula: 10% vs. 0%; and blastocyst: 7% vs. 0%). In conclusion, cow, swamp buffalo, pig, and elephant fibroblasts can be produced by using enucleated rabbit oocytes as recipient cytoplasm. The developmental capacity of cloned embryos is affected by the sources and the individual ability of donor cell used, and varies according to the species.
This study was supported by Ratchadaphiseksomphot Endowment Fund, Reproductive Biotechnology Research Unit, Chulalongkorn University, and Royal Golden Jubilee, PhD program of Thailand Research Fund.
73 RE-CLONING BY SOMATIC CELL NUCLEAR TRANSFER FROM A CLONED KOREAN NATIVE GOAT
Authors: Park J. K. , Jung S. Y. , Park S. H. , Ha A. N. , Jin J. I. , Sohn S. H. , Lee J. S. , Jung J. Y. , Park H. S.
Published: December 2006

Nuclear transfer (NT) is one of the most advanced technologies to increase animal efficiency in livestock production. Re-cloning can be utilized to investigate more effective methods for agricultural, biological, and medical research. However, few studies have been undertaken on re-cloning from cloned animals. The present study was conducted to examine some factors affecting in vitro development of re-cloned embryos and pregnancy by somatic cell nuclear transfer (SCNT). Ear fibroblast cells as karyoplast donors were isolated from a cloned Korean goat, Jinsoonny, 3 weeks after birth and cultured in serum-starvation condition (TCM-199 + 0.5% FBS) for cell confluence. Recipient oocytes were surgically collected by flushing the oviducts at 35 h after hCG injection from FSH-stimulated goats. The zonae pellucidae of the oocytes were partially drilled using a laser system and each somatic cell was individually transferred into an enucleated oocyte. The couplets were electrically fused and activated by ionomycin (5 min) and 6-DMAP (4 h). The reconstructed embryos were cultured in mSOF medium containing 0.8% BSA at 39�C in an atmosphere of 5% CO2, 5% O2, 90% N2 for 12 to 15 h. Re-cloned embryos (2- to 4-cell stages) were surgically transferred into the oviducts of the recipients. Estrous synchronization was induced by 10 days of treatment with a CIDR and a single injection of PGF2�. Pregnancy was diagnosed by progesterone assay and ultrasound on Days 21 and 63 of pregnancy. The fusion and cleavage rates of re-cloned oocytes were 87.5% (182/208) and 56.0% (102/182), respectively. A total of 175 re-cloned embryos were transferred into 28 recipients. Eleven (39.3%) and 4 recipients (14.3%) were confirmed pregnant on Days 21 and 60, respectively. In comparison of pregnany rate by estrous synchronization, a total of 66 and 109 re-cloned embryos were transferred into 11 recipients in natural estrus and 17 recipients in induced estrus, respectively. Five (45.4%) and 2 recipients (18.2%) in natural estrus were confirmed pregnant on Days 21 and 63, while 6 (35.3%) and 2 (11.8%) recipients in induced estrus were pregnant, respectively. There were no differences in pregnancy rate when the recipients were in estrus within 12 h of the donors (40 to 60%). However, the pregnancy rate was significantly decreased when the difference was greater than 24 h (0 to 35%; P << 0.05). Re-cloning can be used for many purposes, and synchronization between donors and recipients may be an important factor for further success of nuclear transfer.
78 BLIND ENUCLEATION OF OOCYTES IS HIGHLY EFFICIENT IN ZONA-FREE BOVINE CLONING
Authors: Prokofiev M. I. , Stepanov O. I. , Komissarow A. V. , Antipova T. A. , Pinyugina M. V. , Malenko G. P.
Published: December 2006

In animal cloning, recipient oocytes are commonly enucleated in metaphase II (MII). By removal of the first polar body (PB1) and 20–30% of the adjacent cytoplasm, without chromatin staining (blind enucleation), only 60% of bovine oocytes can be enucleated (Bordignon and Smith 1998 Mol. Reprod. Dev. 49, 29–36). It is possible that PB1 displaces relative to the MII spindle during removal of cumulus cells (Mohamed Nour and Takahashi 1999 Theriogenology 51, 661–666; Dominko et al. 2000 Biol. Reprod. 62, 150–154). The present study examined the efficiency of blind enucleation of oocytes during preparation of ooplasts for zona-free bovine cloning. Abattoir-derived bovine oocytes were maturated in TCM-199. At 16 h post-maturation (hpm), cumulus cells were removed by vortexing. The zona pellucida (ZP) was removed with pronase. Oocytes with PB1 were retained for enucleation. The rest of oocytes were returned to the maturation medium and checked at an interval of 30–60 min for PB1 extrusion. PB1 together with a minimal volume of adjacent cytoplasm was aspirated using a micromanipulator-controlled micropipette (perpendicular broken, 25–30 �m in diameter). After enucleation, presumptive ooplasts and aspirated oocyte fragments with PB1 were stained with Hoechst 33342. The MII spindle location was determined in UV light under a microscope. In 5 experiments, 227 presumptive ooplasts were investigated. By means of blind enucleation, chromatin was eliminated from 220 oocytes. Maternal chromosomes were found in the cytoplasm of only 7 oocytes after enucleation. The success rate of blind enucleation of bovine zona-free oocytes amounted to 96.9% (220/227). Since the PB1 is fixed on the surface of the zona-free oocyte, when chromosomes migrating with the PB1 are still attached to the microtubule of the spindle, it is a clearly discernible and accurate indicator of maternal chromosome location. The volume of the oocyte cytoplasm removed with the MII plate was about 3.0%. Removal of cumulus cells at 15-16 hpm does not decrease the degree of nuclear maturation of oocytes and has no effect on the development of parthenogenetic or cloned embryos (Galli et al. 2002 Cloning Stem Cells 4, 189–196; Li et al. 2006 Mol. Reprod. Dev. 73, 446–451). In our experiments, the ooplasts prepared by the method of blind enucleation of oocytes without ZP ensured the development to the blastocyst stage from 30 to 50% of reconstructed bovine embryos when fetal fibroblasts were used as nuclear donors. The data obtained suggest that blind enucleation of oocytes is a simple and efficient method to prepare high-quality recipient ooplasts for zona-free bovine cloning.
79 EFFECTS OF EPIDERMAL GROWTH FACTOR, β-MERCAPTOETHANOL, GLUCOSE, OXYGEN CONCENTRATION, AND FIBROBLAST SUBCULTURE ON THE DEVELOPMENT OF PORCINE NT EMBRYOS
Authors: Seok H. B. , Quan J. H. , Kim S. K.
Published: December 2006

The purpose of this study was to investigate in vitro maturation rate of oocytes cultured in maturation medium supplemented with epidermal growth factor (EGF), β-mercaptoethanol (ME), and glucose, and the further development of NT embryos under various conditions. The basic media used for oocyte maturation were NCSU-23 and PZM-3 supplemented with 0.1 mg mL-1 cysteine, 10% (v/v) porcine follicular fluid (pFF), 10 �g mL-1 FSH, 10 �g mL-1 LH, 20 ng mL-1 EGF, and 25 �M ME. Porcine ovaries were collected at a local slaughterhouse, and donor cells from a 35-day-old fetus were dissociated, resuspended, and cultured for 6–8 days in DMEM supplemented with 10% (v/v) FBS, penicillin G (75 �g mL-1), streptomycin (50 �g mL-1), 1 mM sodium pyruvate, and 1% (v/v) nonessential amino acids. The first polar body and adjacent cytoplasm were enucleated by a micropipette in HEPES-buffered NCSU-23 supplemented with 4 mg mL-1 BSA and 7.5 �g mL-1 cytochalasin B. Couplets were equilibrated with 0.3 M mannitol solution and transferred to a chamber containing 2 electrodes with a pulse of 2.1 kV cm-1 for 30 �s. When the embryos were cultured in NCSU-23 or PZM-3 supplemented with or without 20 ng mL-1 EGF for 144 h, the development rates to the blastocyst stage were 12.0 � 1.3%, 9.6 � 1.9%, 10.9 � 2.1%, and 9.1 � 2.3%, respectively. When the embryos were cultured in NCSU-23 or PZM-3 supplemented with or without 25 �M ME for 144 h, the rates to blastocyst stage were 9.6 � 1.7%, 7.3 � 2.3%, 11.9 � 1.8%, and 7.4 � 2.1%, respectively. The developmental rate to the blastocyst stage of NT embryos cultured in PZM-3 supplemented with ME was significantly higher than when cultured without ME supplementation (P < 0.05). When the embryos were cultured in NCSU-23 or PZM-3 supplemented with or without 1.5 mM glucose for 144 h, the rates to blastocyst stage were 9.4 � 2.2%, 6.8 � 2.7%, 10.9 � 2.4%, and 8.9 � 2.6%, respectively. The developmental rate to the blastocyst stage of NT embryos cultured in NCSU-23 and PZM-3 supplemented with glucose was higher than when cultured without glucose supplementation. When NT embryos were cultured in NUSU-23 and PZM-3 at 5% and 20% O2 concentration, the rates were 11.1 � 1.8%, 9.8 � 1.4%, 12.5 � 1.6%, and 10.9 � 1.5%, respectively. The developmental rate to the blastocyst stage of NT embryos cultured in both NCSU-23 and PZM-3 at 5% O2 concentration was higher than when cultured at 20% O2 concentration. When fetal fibroblasts were cultured in NCSU-23 and PZM-3, the fusion rate of less than 10 passages was higher than for those of 11–15 passages. In conclusion, the present study indicates that EGF and glucose have beneficial effects on the in vitro maturation of oocytes, and ME improves the developmental ability of NT embryos. Furthermore, the developmental rate in subcultured fibroblast cells was improved when reconstruction was made with less than 10 passages.
8 PREGNANCY RATE FOLLOWING TIMED AI IN BEEF HEIFERS TREATED WITH CUE-MATE AND pLH OR GnRH
Authors: Colazo M. G. , Dias F. C. , Lightfoot K. , Dochi O. , Kastelic J. P. , Mapletoft R. J.
Published: December 2006

The objective was to investigate the use of Cue-Mate and porcine LH (pLH) or GnRH with or without presynchronization on pregnancy rate following timed AI (TAI) in Angus heifers (n = 462). Approximately half of the heifers (Control; n = 236) were treated at random stages of the estrous cycle, and the other half (Presynch; n = 226) received two injections of 500 �g cloprostenol (PGF: Estrumate�; Schering Plough Animal Health, Pointe-Claire, Quebec, Canada) 14 days apart; synchronization treatments were initiated 11 days after the second injection of PGF. On Day 0, heifers received an intravaginal progesterone device (Cue-Mate; Bioniche Animal Health, A/Asia Pty, Armidale, Australia) and were treated with 100 �g GnRH IM (Cystorelin�; Merial Canada Inc., Victoriaville, Quebec, Canada; n = 233) or 12.5 mg pLH (Lutropin-V�; Bioniche; n = 229). On Day 7, Cue-Mates were removed and heifers were given PGF IM. GnRH or pLH (same as the first treatment) was given concurrently with TAI on Day 9 (54–56 h after PGF) with frozen–thawed semen of one of 3 sires. Ultrasonographic examinations were performed in a subset of 182 heifers on Days 0 and 7 for CL and follicle development, and in all heifers on Days 41 to 49 for confirmation of pregnancy. Data were compared using CATMOD procedures in the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). The proportion of cycling heifers on Day 0 was 90.7% (165/182). Pregnancy rate tended to differ among bulls (46.7, 56.9, and 61.4% for Bulls A, B, and C, respectively; P≤ 0.1). Heifers that ovulated in response to the first GnRH or pLH injection had a higher pregnancy rate than those that did not ovulate (66.3 vs. 51.9%; P ≤ 0.03). In addition, heifers treated with GnRH tended to have a higher ovulatory response to the first treatment and a higher pregnancy rate to TAI than those treated with pLH (60.9 and 60.5% vs. 50.0 and 52.4%, respectively; P ≤ 0.09). Although ovulatory response to the first GnRH or pLH treatment was 46.5 and 63.5% for Control and Presynch groups, respectively (P ≤ 0.02), pregnancy rates did not differ (59.8 vs. 53.1%; P ≥ 0.2). However, there was an interaction between presynchronization treatment and ovulatory response to the first injection of GnRH or pLH on pregnancy rates (P ≤ 0.02). The pregnancy rate was higher in Control heifers that ovulated (77.5%) to the first injection of GnRH or pLH than in Control heifers that did not ovulate (52.2%) or Presynch heifers that did (59.0%) or did not (51.4%) ovulate. In summary, Cue-Mate-treated heifers that ovulated in response to the first GnRH or pLH treatment had higher pregnancy rates to TAI. Although presynchronization with PGF increased the ovulation rate, it did not significantly affect the pregnancy rate.
80 EFFECT OF MEDIA, SYNCHRONIZATION OF FIBROBLAST CELLS, CULTURE TIME, OXYGEN CONCENTRATION, AND ACTIVATION ON NUCLEAR-TRANSFERRED PORCINE OOCYTES
Authors: Quan J. H. , Seok H. B. , Kim S. K.
Published: December 2006

The purpose of this study was to investigate the impact of culture medium, culture duration, and atmospheric condition on the fusion and in vitro development rates of nuclear transfer porcine embryos constructed by the microinjection of fetal fibroblast cells into in vitro-matured oocytes. Single fetal donor cells were deposited into the perivitelline space of enucleated oocytes, followed by electrical fusion and activation. Activated embryos were cultured in NCSU-23 medium supplemented with 5% FBS, at 38.5�C for 6 to 8 days in 5% CO2 and air. In Experiment 1, the fusion rates of nuclear transfer embryos did not differ from those of fetal fibroblast cells incubated in 5% FBS + NCSU-23 or 5% FBS + TL-HEPES medium, nor did fusion rates of donor cells differ among 1–8-h incubation durations. Fusion rates for the 4 treatment subclasses ranged from 72.1% to 78.0%. In Experiment 2, pre-synchronization in medium containing 0.1 �g mL-1 Hoechst(H) 33342 increased during the period from 0 and 8 h of culture up to 15 h, the end of the synchronization period, at which time there was a significantly increased percentage of porcine fibroblast cells at the G2/M stage (12.4%, 17.5%, and 47.6%; P < 0.01). Neither an increase in the concentration of H 33342 (0.2–1.6 �g mL-1) nor a longer exposure time (12 h, 18 h, and 24 h) increased the proportion of porcine G2/M fibroblasts. In Experiment 3, fusion rates did not differ significantly between nuclear transfer embryos constructed using donor cells cultured in 5% FBS + NCSU-23 medium for 1–2, 6–8, or 12–14 days (60.0%, 73.3%, and 62.5%, respectively). The cleavage rate for nuclear transplant embryos using fetal fibroblast cells cultured for 1–2 days was 44.0%, which was significantly less than the 56.7% and 50.0% for 6–8 or 12–14 days of culture, respectively (P < 0.05). In Experiment 4, the proportions of nuclear transfer embryos that developed to the e2 cell and to the blastocyst stage were not affected significantly by culture medium (5% FBS + NCSU-23 or 5% FBS + TL-HEPES) or by O2 concentration during culture (5% vs. 10%). The developmental rates to the e2 cell stage ranged from 65.9% to 70.1%, and those to the blastocyst stage ranged from 9.8% to 12.5%, for the 4 treatment subclasses. Blastocyst rate was highest for embryos cultured in 5% FBS + NCSU-23 under a gas atmosphere of 5% O2 in air.
82 EFFECT OF DONOR CELL TRANSFECTION EVENTS ON EMBRYO AND FETAL SURVIVAL IN CLONING
Authors: Salamone D. F. , Santos C. B. , Barañao J. L. , Bussmann L. , Artuso J. , Munar C. J. , Berra G. , Criscuolo M. , Melo C.
Published: December 2006

In a large-scale bovine cloning program intended to obtain transgenic animals, it is important to maximize the number of calves produced. The present experiment was designed to test the hypothesis that different transfection events of the same somatic cell line can affect embryo and/or fetal survival. A fetal cell line was established from a 75-day-old Jersey female fetus. It was used as control and was also transfected 3 times with the same protocol. They were named Transfection 1, 2, and 3. Genetically modified cells were produced and isolated after selection with geneticin for 10–15 days following liposome transfection with a DNA construct containing a selectable neomycin resistance gene. Oocytes were aspirated from slaughterhouse ovaries and matured in TCM-199 + 5% FCS at 39°C for 24 h. Matured oocytes were denuded by vortexing for 3 min in TL-HEPES with 1 mg mL−1 bovine testis hyaluronidase. Metaphases were assessed, and oocytes were enucleated by visualization with Hoechst 33342 (5 µg mL−1) under UV light (<6 s). Donor cells from different treatments were used for nuclear transfer at G0/G1 cell cycle stages and were fused to enucleated oocytes by an electrical pulse. After 3 h, activation was induced by incubation in TL-HEPES with 5 µM ionomycin for 4 min and 2 mM 6-DMAP for 3 h. The oocytes were then washed with TL-HEPES and cultured in SOF medium with an atmosphere of 5% CO2 + 5% O2 + 90% N2. Development to blastocysts (Days 7 to 9) was recorded. Two blastocysts were transferred nonsurgically per recipient cow, and pregnancies at 30 days were determined by ultrasonography. All data were analyzed by chi-square test. In vitro development to blastocysts was similar in all treatment groups. One birth was obtained from the control. Four and 7 births were obtained from Transfections 1 and 3, respectively. Although Transfection 2 had good in vitro development, this treatment did not produce any pregnancy. This fact demonstrated that the transfection event provides an additional source of variability in obtaining live transgenic animals. Our results pointed out the necessity to monitor fetal survival by ultrasonography in order to detect as soon as possible any deficiencies in development introduced by transfection.

Table 1.Effect of different transfection events of same line on embryo and fetal survival
83 IN VITRO DEVELOPMENTAL ABILITIES OF PORCINE CLONED EMBRYOS RECONSTITUTED WITH CELL NUCLEI OF GENETICALLY TRANSFORMED FETAL FIBROBLASTS CYTOMETRICALLY DIAGNOSED ON CELL CYCLE AND APOPTOSIS
Authors: Samiec M. , Skrzyszowska M. , Bochenek M. , Lipinski D. , Slomski R.
Published: December 2006

The important factor that determines the development of mammalian cloned embryos is structuro-functional quality of nuclear donor cells. Analysis of nuclear DNA (nDNA) content of somatic cells undergoing apoptosis has become one of the most common methods for single-parameter flow cytometric measurement of this process. Apoptosis assessment is performed by quantification of hypodiploid cells. The aim of our study was to examine the in vitro developmental potential of porcine nuclear transfer (NT) embryos reconstituted with non-apoptotic fetal fibroblast cells expressing the eGFP transgene. The nuclear donor cells were derived from cell line populations whose representative random samples had been analyzed on both cell cycle and apoptosis through non-vital nDNA fluorescent dyeing and flow cytometry (FACS). Frozen-thawed fibroblast cells, which had been cultured up to a total confluency after 2–4 passages, were used for the diagnostics. The cells were fixed in ice-cold 70% ethanol. Then, the fetal fibroblasts were exposed to nDNA extraction buffer for 5 min at room temperature, and incubated in DNA staining solution (propidium iodide and RNAse) for 30 min. After fluorescent labeling, the cells were analyzed in the flow cytometer by reading nDNA fluorescence in the red band. In vitro-matured oocytes were the source of recipient cells. Fibroblast cell–ooplast couplets were simultaneously fused and activated. Reconstructed embryos were cultured in NCSU-23/BSA/FBS medium for 6–7 days. The rates of cleavage and development to morula/blastocyst stages were examined on Days 2 and 6/7, respectively. FACS analysis revealed that, out of all of the diagnosed fetal fibroblast cells, 54.7% were cycling, and up to 45.3% were late-apoptotic. In turn, from among the normal (i.e. non-apoptotic) cells, 82.2% were at G0/G1 stages of cell cycle, 17.0% at the S stage, and 0.8% at G2/M stages. A total of 150 enucleated oocytes were successfully fused with non-apoptotic transgenic nuclear donor cells. Out of 150 cultured NT embryos, 123 (82.0%) were cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages yielded 53/150 (35.3%) and 37/150 (24.7%), respectively. In conclusion, the FACS analysis for mitotic cycle of 100%-confluent transgenic fetal fibroblasts confirmed the high efficiency of the cell cycle synchronization at G0/G1 phases. However, a contact inhibition method induced the high frequency of late-apoptotic cells. Moreover, the relatively high percentage of NT blastocysts was developed from oocytes reconstructed with eGFP transgenic fetal fibroblast cells.
This research was supported by the State Committee for Scientific Research as a Solicited Project number PBZ-MIN-005/P04/2002/6 from year 2003 to year 2006.
90 GERM-LINE TRANSMISSION OF DONOR MITOCHONDRIAL DNA IN NUCLEAR TRANSFER-DERIVED COWS
Authors: Takeda K. , Kaneyama K. , Tasai M. , Akagi S. , Yonai M. , Miyashita N. , Onishi A. , Tagami T. , Nirasawa K. , Hanada H.
Published: December 2006

In embryos derived by nuclear transfer (NT), fusion, or injection of donor cells with recipient oocytes caused mitochondrial heteroplasmy. Previous studies have reported varying patterns of mitochondrial DNA (mtDNA) transmission in cloned calves. Distribution of donor mtDNA found in offspring of NT-derived founders may also vary from donor–host embryo heteroplasmy to host embryo homoplasmy. Here we examined the transmission of mtDNA from NT cows to their progeny. NT cows were originally produced by fusion of enucleated oocytes with Jersey (J) or Holstein (H1) oviduct epithelial cells, or Holstein (H2) or Japanese Black (B) cumulus cells, as previously reported (Goto et al. 1999 Anim. Sci. J. 70, 243–245; Yonai et al. 2005 J. Dairy Sci. 88, 4097–4110; Akagi et al. 2003 Mol. Reprod. Dev. 66, 264–272). Transmission of donor cell mtDNA was analyzed by PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis of the mitochondrial D-loop region. Eleven NT founder cows were analyzed, 4 (2 = J-NT, and 2 = H1-NT) of them were heteroplasmic whereas 7 (1 = J-NT, 1 = H1-NT, 2 = H2-NT, and 3 = B-NT) were homoplasmic for the host embryo mitochondria. The proportions of donor mtDNA detected in one J-NT cow was 7.7%, and those of other cow lineages were <2%. Heteroplasmic NT cows delivered a total of 9 progeny. Four of the 9 progeny exhibited heteroplasmy with high percentages of donor cell mtDNA populations (52%, 37%, 17%, and 43%). The other 5 progeny were obtained from heteroplasmic NT cows, and all samples of the 10 progeny obtained from the homoplasmic NT cows did not harbor detectable donor cell mtDNA. A genetic bottleneck in the female germ-line will generally favor the transmission of a single mitochondrial population, leading to a return to homoplasmy. Thus, some of progeny maintained heteroplasmy with a higher ratio than that of their NT mothers, which may also reflect a segregation distortion caused by the proposed mitochondrial bottleneck. These results demonstrated that donor mtDNA in NT cows could be transmitted to progeny with varying efficiencies, in a lineage-specific fashion.
97 CLONED CALF PRODUCTION BY AGGREGATION OF SOMATIC CELL NUCLEAR-TRANSFERRED EMBRYOS
Authors: Yamaguchi D. , Suzuki W. , Adachi N. , Akagi S. , Watanabe S. , Takahashi S.
Published: December 2006

A low pregnancy rate and high frequency of abortion are observed in bovine somatic cell cloning. It is suggested that one of the factors is the low cell number of nuclear-transferred (NT) embryos at the blastocyst stage compared with that of in vivo-derived embryos. We reported that aggregation of bovine NT embryos can develop to blastocysts with cell numbers that are equivalent to in vivo-derived embryos (Akagi et al. 2005 Reprod. Fertil. Dev. 17, 162 abst). In this study, we examined the in vivo development of aggregates of NT embryos after embryo transfer (ET). Following culture in serum-starved medium for 5 to 7 days, fibroblast cells were used as donor cells for NT. NT was performed as previously described (Akagi et al. 2003 Mol. Reprod. Dev. 66, 264–272). Eight-cell stage embryos on Day 2 or 16- to 32-cell stage embryos on Day 4 were used for embryo aggregation after removal of the zona pellucida. Three NT embryos were placed into the depression in a drop of TCM-199 with 50 µg mL−1 phytohemagglutinin for 20 min, and NT aggregates were then moved into the depression in a drop of IVD-101 (Akagi et al. 2005). On Day 7 after NT, ET was performed nonsurgically. Results are summarized in Table 1. Pregnancy rates obtained with the aggregated NT embryos tended to be high compared with the single NT embryos. All pregnant cows in the single NT embryos and 6 of 8 pregnant cows in the aggregated NT embryos were aborted within 90 days. One cow carrying an aggregate of 3 Day 2 NT embryos went to term, and one carrying an aggregate of 3 Day 4 NT embryos is still pregnant at more than 7 months. These results demonstrate that aggregates of 3 NT embryos can develop to term in cattle.

Table 1.In vivo development of aggregated NT embryos
98 BLASTOCYST DEVELOPMENT RATE OF CLONED CAT EMBRYOS USING SERIAL CLONING
Authors: Yin X. J. , Lee H. S. , Choi E. G. , Yu X. F. , Choi B. H. , Piao M. S. , Oh D. H. , Lee S. S. , Kong I. K.
Published: December 2006

Domestic cats are a useful research model to develop assisted reproductive technologies for the conservation of endangered felids. Previously, we produced cloned offspring derived from somatic cell nuclear transfer of ear skin fibroblasts obtained from a deaf, odd-eyed, male Turkish Angora. The aim of this study was to assess the cloning efficiency of the fibroblasts derived from a cloned cat. Fibroblast cell lines were established from 6-mm skin biopsies taken from a deaf, odd-eyed, male Turkish Angora and his clone. The protocol for nuclear transfer was described previously (Yin et al. 2005 Reproduction 129, 245–249). Briefly, cumulus cells were removed from the ova by gently pipetting them into TCM-199 supplemented with 0.1% hyaluronidase. The denuded oocytes were then cultured in TCM-199 supplemented with 0.2 �g mL-1 demecolcine for 1 h and placed into TCM-199 containing 5 �g mL-1 cytochalasin B and 0.2 �g mL-1 demecolcine. The first polar body and protruded chromatin plate were removed with a beveled micropipette. Micromanipulation was used to place a single donor cell nucleus into the perivitelline space of enucleated ova. The ovum-cell couplets were fused and pulse activated. The activated couplets were cultured in 500 �L of CRI medium supplemented with 0.3% BSA for 2 days. The cleaved embryos were cultured in CRII medium supplemented with 10% FBS for 5 days. The cleavage and blastocyst development rates were 38.5% and 3.5% for second generation cloned embryos. A total of 310 second generation cloned embryos were transplanted to 9 surrogates, and 2 pregnancies at 30 days were determined by ultrasonography. One pregnancy was aborted at 40 days of gestation; the second pregnancy continued. These results indicate that the serial cloning of a cat can be generated efficiently up until pregnancy.
This work was supported by KOSEF (grant #M10525010001-05N2501-00110).
Maternal serotonin is crucial for murine embryonic development
Authors: Côté Francine , Fligny Cécile , Bayard Elisa , Launay Jean-Marie , Gershon Michael D. , Mallet Jacques , Vodjdani Guilan
Published: December 2006

The early appearance of serotonin and its receptors during prenatal development, together with the many effects serotonin exerts during CNS morphogenesis, strongly suggest that serotonin influences the development and maturation of the mammalian brain before it becomes a neuromodulator/neurotransmitter. Sites of early serotonin biosynthesis, however, have not been detected in mouse embryos or extraembryonic structures, suggesting that the main source of serotonin could be of maternal origin. This hypothesis was tested by using knockout mice lacking the
tph1
gene, which is responsible for the synthesis of peripheral serotonin. Genetic crosses were performed to compare the phenotype of pups born from homozygous and heterozygous mothers. Observations provide the first clear evidence that (
i
) maternal serotonin is involved in the control of morphogenesis during developmental stages that precede the appearance of serotonergic neurons and (
ii
) serotonin is critical for normal murine development. Most strikingly, the phenotype of
tph1
−/− embryos depends more on the maternal genotype than on that of the concepti. Consideration of the maternal genotype may thus help to clarify the influence of other genes in complex diseases, such as mental illness.