bluuebunny/crossref_metadata_2025 huggingface dataset:
386 EFFECTS OF TIME AND NUMBER OF INSEMINATIONS ON THE SEX RATIO OF EMBRYOS IN SUPERSTIMULATED HOLSTEIN HEIFERS
Authors: Lim K. T. , Ideta A. , Lee W. Y. , Jung Y. G. , Hayama K. , Urakawa M. , Aoyagi Y.
Published: December 2006

It is necessary to control the sex ratio (percentage of females) of pre-implantation embryos in the dairy industry. Several studies indicate that, in cattle, varying insemination time does not influence the embryo sex ratio (Rorie et al. 1999 Theriogenology 52, 1273–1280). However, it has been suggested that insemination early in estrus would result in more females, and late in estrus, more males, due to different timing of capacitation and survival of X- and Y-chromosome-bearing spermatozoa in the female reproductive tract (Martinez et al. 2004 Theriogenology 62, 1264–1270). The objective of the present study was to examine whether the time of insemination affects the sex of 7-day embryos in superstimulated Holstein heifers. Donor heifers (14 to 18 months of age, n = 38) were given decreasing doses of FSH IM (total of 28 Armour units, Antrin R-10; Kawasaki-Mitaka, Kanagawa, Japan) twice daily for 4 days between Days 8 and 10 of the estrous cycle. Five (25 mg) and 3 (15 mg) mL of dinoprost (PGF; Veterinary Pronalgon; Pfizer Animal Health, Tokyo, Japan) were administered IM with the seventh and eighth FSH treatments, respectively. The heifers received 50 �g of fertirelin acetate IM (GnRH; Conceral; Schering-Plough Animal Health KK, Tokyo, Japan) 48 h after the first PGF treatment. Heifers were inseminated with 2 straws of frozen–thawed semen into the uterine body at 48 h (n = 18), 60 h (n = 10), or 60 + 72 h (n = 10; control) after the first PGF treatment. In this study, frozen–thawed semen from several bulls was used. Seven-day embryos were recovered by uterine flushing. Grade 1 embryos (IETS classification) were selected for male and female separation using the loop-mediated isothermal amplification procedure. The data (mean � SEM) were compared with Fisher's protected least significant difference test following ANOVA. The mean number of ova/embryos recovered was 12.1 � 1.9 (48 h), 12.8 � 2.8 (60 h), and 11.0 � 1.6 (60 + 72 h). There were no significant differences in the percentages of unfertilized ova (48 h: 6.9 � 2.1%, 15/218; 60 h: 7.8 � 3.2%, 17/128; and 60 + 72 h: 10.0 � 3.6%, 11/111; P > 0.05) and in grade 1 embryos (48 h: 61.2 � 4.9%, 124/218; 60 h: 64.9 � 7.9%, 65/128; and 60 + 72 h: 59.1 � 4.8%, 66/111; P > 0.05) among groups. Furthermore, the female ratio of grade 1 embryos was not significantly different for the 3 insemination times (48 h: 50.0 � 5.8%, 56/124; 60 h: 48.4 � 5.0%, 33/65; and 60 + 72 h: 52.2 � 8.0%, 36/66; P > 0.05). These results indicate that a single early insemination during estrus does not reduce the fertilization rate and embryo quality in superstimulated Holstein heifers. Also, the time of insemination did not appear to have any effect on the sex ratio of embryos from these heifers.
393 A STUDY OF THE USE OF SEXED SEMEN IN SUPERSTIMULATED HOLSTEIN COWS
Authors: Wang H. , Li R. Q. , Zhang X. L. , Pan Q. Z. , Li J. L. , Zhao M. B. , Wang Y. Z. , Chen D. Y.
Published: December 2006

A study was designed to investigate the use of sexed semen in superstimulated Holstein cows. Non-lactating Holstein cows (n = 107; those that did not superovulate were excluded) were superstimulated and inseminated with frozen–thawed, sexed semen (at 1 insemination dose) or control semen (at 2 different insemination doses) in 3 groups. Cows received a Cue-Mate (Bioniche Animal Health, Beijing, China) and were injected with 5 mg of estradiol-17β plus 100 mg of progesterone on Day 0. Cows were treated from Days 4 to 7 with decreasing doses of FSH for a total of 300 mg Folltropin-V (Bioniche; 50, 50, 40, 40, 30, 30, 30, and 30 mg) at 12 h intervals. On Day 7, Cue-Mates were removed and cows were injected twice with 0.4 mg cloprostenol (PGF; Institute of Family Planning, Shanghai, China). Bilateral deep uterine horn AI was performed 12 and 24 h after the onset of estrus on Day 9 (Group A: 4 × 106 sexed, frozen–thawed sperm per insemination from 1 of 3 bulls; Group B: 4 × 106 non-sexed, frozen–thawed sperm, or Group C: 10 × 106 non-sexed, frozen–thawed sperm per insemination from 1 of 3 bulls). Cows were assigned to bulls and insemination groups at random. Embryos were collected and evaluated on Day 16. Group A had a significantly lower fertilization and usable embryo (IETS grades 1 and 2) rate than the other 2 groups (Table 1). Although the rate of usable embryos between Groups B and C did not differ, the fertilization rate in Group C was significantly higher, suggesting that 4 million sperm in Group B was an inadequate insemination dose for superstimulated cattle. The significantly lower number of usable embryos in Group A suggests that the use of sexed sperm in superovulation and embryo transfer may not be economically feasible. More research is required to optimize and standardize bovine superstimulation and AI with sexed semen.

Table 1.
Embryo production following insemination of superstimulated Holstein cows with frozen–thawed sexed (Group A) or unsexed (Groups B and C) semen
395 ISOLATION AND CHARACTERIZATION OF PORCINE ADIPOSE-DERIVED ADULT STEM CELLS
Authors: Picou A. A. , Bartolotta S. L. , Williams K. J. , Giraldo A. M. , Godke R. A. , Bondioli K. R.
Published: December 2006

A dipose-derived adult stem (ADAS) cells have been proposed as an alternative to embryonic stem cells for tissue engineering applications. Stromal cells isolated from human adipose tissue are self-renewing, can be induced to differentiate along several mesenchymal tissue lineages, and are characterized by a high expression of CD34 and CD44 stem cell markers. The objective of this study was to develop a protocol for the isolation and culture of porcine ADAS cells and to determine stem cell-like characteristics such as the ability to differentiate into multiple cell lineages and expression of CD34 and CD44. Ten grams of subcutaneous fat were collected from mature pigs, finely minced, and washed with PBS. The tissue was homogenized with 1% collagenase in PBS with 0.1% bovine serum albumin in a shaker incubator at 37�C for 2 h. The cell suspension was filtered and stained with Hoechst 33342 to determine the number of nucleated cells in the suspension. Adherent cells were cultured in DMEM supplemented with 10% fetal bovine serum and incubated at 39�C in 5% CO2. At 90% confluence, cells were passaged by trypsinization and cultured for 21 days in lineage-specific cocktails previously demonstrated to induce differentiation. Stains specific for intracellular lipids, glycosaminoglycans, and calcium deposition were used to detect adipocyte, osteocyte, and chondrocyte differentiation, respectively. mRNA from undifferentiated and differentiated cells was isolated using a Dynabeads mRNA Kit (Dynal, Lake Success, NY, USA), and cDNA was synthesized using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). RT-PCR was performed with primers for Poly A Polymerase (PAP; reference gene), CD34, and CD44. PCR reaction products were subjected to electrophoresis and analyzed by Quantity One software (Bio-Rad). An average of 2 300 000 nucleated cells/mL was isolated from the fat sample; 26% of the cells were adherent, and the cells completed a cell cycle approximately every 2.8 days. After culture in differentiation medium, 57% of cultures stained positive with Nile red (adipocytes), 87% stained with Safranin O (chondrocytes), and 50% stained with Alizarin Red (osteocytes). The number of pixels in each electrophoresis band was used to determine the relative levels of gene expression and is represented as a ratio of CD34 or CD44 to PAP. In undifferentiated cells, the band intensity ratio for CD34 : PAP was 1.54 and that for CD44 : PAP was 0.96. Ratios for differentiated cells were as follows (CD34 : PAP, CD44 : PAP): adipocytes (0.38, 1.1), chondrocytes (0.24, 1.0), osteocytes (0.63, 0.99), and fibroblasts (0.38, 1.14). The CD34 expression level was lower than that of CD44 among the differentiated cells. CD34 expression was higher for ADAS compared with differentiated cells; however, CD44 did not follow this pattern. CD34 but not CD44 expression appears to be a good marker for differentiation in these cells.
402 OOCYTE-MEDIATED GENE TRANSFER: A NOVEL APPROACH TO PRODUCE TRANSGENIC PORCINE EMBRYOS
Authors: Gupta M. K. , Uhm S. J. , Kim E. Y. , Jung Y. H. , Yu J. Y. , Lee H. T.
Published: December 2006

Classical approaches for producing transgenic livestock require labor-intensive, time-consuming, and expensive methods but have low transgenic efficiency and a high mosaicism rate. This study evaluated a simplified method for producing transgenic porcine embryos by microinjecting a DNA construct into unfertilized metaphase oocytes that were subsequently fertilized in vitro. For this, oocytes recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro for 42–44 h and were microinjected with DNA solution (10 ng �L-1) using a femtojet microinjector (Eppendorf, Hamburg, Germany). The DNA (4.7 kb) was derived from the pEGFP-C1 plasmid (Clontech Laboratories Inc., Palo Alto, CA, USA), which contains the enhanced green fluorescent protein (EGFP) encoding transgene under the control of cytomegalovirus promoter, and linearized with ApaLI restriction enzyme. Injected oocytes were then in vitro-fertilized using fresh epididymal sperm obtained from abattoir-derived porcine testis by standard procedure and cultured in NSCU23 medium supplemented with 0.4% BSA. The efficiency of transgenesis was monitored by visualization of green florescence under UV illumination using a EGFP filter set. Data were analyzed by Student's t-test. Results showed that the cleavage rate of injected oocytes (68.7 ± 0.5%) was similar to that of non-injected control oocytes (67.8 ± 0.4%). However, a high percentage of injected oocytes showed a developmental block at the 2–4 cell stage. The EGFP expression rate at 2–4 cell stage, when expressed as proportion of injected oocyte, was 17.2 ± 0.1%. Interestingly, mosaicism was not observed. The EGFP expression rate increased to 26.7 ± 0.1% when the DNA concentration was increased to 40 ng µL−1. Injecting the DNA solution near the metaphase plate of the oocyte did not improve (P < 0.05) the EGFP expression rate (22.2 ± 0.1%). A high proportion of EGFP-expressing oocytes blocked at the 4–8 cell stage and did not progress to blastocyst, possibly due to random integration of the transgene in developmentally important gene loci. Our results thus suggest oocyte-mediated gene transfer as a promising tool for producing transgenic livestock. However, further research is required to improve its efficiency.
This work was supported by the Research Project on the Production of Bio-Organs (No. 200503030201), Ministry of Agriculture and Forestry, Republic of Korea.
403 IDENTIFICATION AND CHARACTERIZATION OF A NOVEL MOUSE AND HUMAN MOPT GENE CONTAINING MORN MOTIF PROTEIN IN TESTIS
Authors: Kim K. M. , Choi Y.-J. , Song H. , Hwang K.-C. , Kang S.-J. , Cho S.-K. , Kim M.-O. , Kim J.-H.
Published: December 2006

By subtraction screening methods, we identified a novel mouse and its counterpart, human MOPT gene. The mouse and human MOT gene is localized on mouse chromosome 17E3 and human chromosome 2p22, and spans approximately 7 kb. Analysis of the mouse Mopt sequence revealed the existence of an ORF of 240 bp encoding a putative protein of 79aa amino acids. The predicted protein has a theoretical molecular mass of 8.89 kDa and a calculated isoelectric point of 5.82. The protein was unique; it did not show any similarities with other known protein except for Morn motif domain. Real-time reverse transcriptase polymerase chain reaction and Northern blot analysis revealed that mouse Mopt transcripts are highly and specifically expressed in adult testis and skeletal muscle. In situ hybridization and immunohistochemistry studies showed that mouse Mopt transcript and protein was confined mainly to round and elongated spermatids, except for a few individual dispersed spermatocytes, and increases in abundance in subsequent stages. To characterize Mopt functions in spermiogenesis, we examined Mopt protein distribution in late spermiogenesis by using immunogold electron microscopy: Mopt protein first appeared in the proacrosomic vesicles of the early Golgi phase spermatids. In the final step of spermiogenesis, Mopt expression was translocated from the head cap of an elongated spermatid to the nucleus of mature spermatozoa. However, no other testicular cell types, including somatic cells, spermatogenic cells, and residual cytoplasm that ultimately is engulfed as residual bodies into Sertoli cells, were found to bear Mopt-positive staining. This observation suggested that Mopt may play an important role in dynamic regulation of acrosome biogenesis during late spermiogenesis and an as yet uncharacterized role in oocyte activation during capacitation, after fertilization, or both.
44 NUCLEAR TRANSFER IN CATTLE USING SOMATIC CELLS FROM FROZEN TESTICLES WITHOUT CRYOPROTECTANTS
Authors: Hoshino Y. , Kobayashi N. , Hayashi N. , Matsuhashi T. , Saeki K. , Ikeda S. , Taniguchi S. , Kasamatsu A. , Iwamoto D. , Abe Y. , Matsumoto K. , Hosoi Y. , Iritani A.
Published: December 2006

Obtaining somatic cells from preserved organs or tissues is useful for the conservation and regeneration of genetic resources by nuclear transfer (NT). Bovine cells for NT have been obtained from cooled carcasses stored at 0�C for several days (Arat et al. 2005 Reprod. Fert. Dev. 17, 164 abst) and from fetal skin tissue cryopreserved with DMSO (Fahrudin et al. 2001 J. Vet. Med. Sci. 63, 1151–1154). However, frozen storage of organs or tissues without cryoprotectants was considered to be quite inappropriate for obtaining viable cells. We report here that viable donor cells for NT were obtained from bovine testicles after frozen storage without cryoprotectants. In the first experiment, we investigated whether viable cells can be recovered from frozen testicles castrated from Japanese Black bulls. The testicles were frozen at -80�C in a freezer for several days; then some were stored in liquid nitrogen for 10 months without cryoprotectants. Before thawing, the testicles were divided into 3 pieces, caput epididymis, cauda epididymis, and testis. Each piece was then put in saline at 42�C for quick thawing. Thawed tissues were minced into 5-mm pieces and incubated at 39�C for 2 h in DMEM containing 0.1% collagenase and 0.2% dispase. After filtration through a 250-�m nylon mesh filter, the filtrates were centrifuged at 250 � 4g for 5 min. Then precipitates were resuspended with MF-start� primary culture medium (TM Cell Research Inc., Fukui, Japan) and incubated at 38.5�C under the atmosphere of 5% CO2 in air with high humidity. After 5 days of incubation, the medium was replaced and nonadherent debris was discarded. Viable cells were obtained from the caput epididymis. These cells actively proliferated and expanded. In the next experiment, to determine whether these cells can be used for NT, the cells were electrically fused with enucleated bovine oocytes. Bovine fibroblasts taken from unfrozen ear tissue were used as controls. The NT embryos were activated by Ca-ionophore treatment, followed by treatment with cycloheximide for 6 h, and then cultured in mSOF for 168 h. NT embryos reconstructed from testicle cells did not significantly differ from NT embryos made with control cells with regard to blastocyst rates (22.1% and 20.2%), cell number of blastocysts [130 � 43 and 121 � 43 (mean � SD)], and ICM ratio (21.1% and 22.6%), respectively (ANOVA). These results suggest that somatic cells derived from bovine frozen testicles can be used for nuclear transfer. Further studies are needed to examine whether viable cells can be obtained from other frozen organs or tissues.
This study was partially supported by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, JST.
47 DIFFERENTIAL DEVELOPMENTAL ABILITY OF EMBRYOS CLONED FROM TISSUE-SPECIFIC STEM CELLS
Authors: Inoue K. , Ogonuki N. , Miki H. , Noda S. , Inoue S. , Katayama K. , Mekada K. , Miyoshi H. , Ogura A.
Published: December 2006

Although cloning animals by somatic cell nuclear transfer is generally an inefficient process, use of appropriate donor cell types may improve the cloning outcome significantly. Among the donor cells tested so far, mouse embryonic stem cells have given the best efficiency in terms of the development of reconstructed embryos into offspring. In this study, we examined whether 2 in vitro-produced pluripotent stem cells—neural stem cells (NSCs) and mesenchymal stem cells (MSCs)—could be better nuclear donors than other differentiated cells. Embryos were reconstructed by transfer of nuclei from NSCs or MSCs with full potential for differentiation in vitro. Most (76%) of the 2-cell NCS embryos developed to the 4-cell stage; 43% implanted and 1.6% developed to term after transfer to pseudopregnant recipients. These rates were very similar to those of embryos cloned from fibroblast cell nuclei. Interestingly, in the patterns of zygotic gene expression, NSC embryos were more similar to in vitro-fertilized embryos than fibroblast cloned embryos. By contrast, embryos reconstructed using MSC nuclei showed lower developmental ability and no implantation was obtained after embryo transfer. Chromosomal analysis of the donor MSCs revealed very high frequencies of monosomy and trisomy, which might have caused the very poor post-implantation development of embryos following nuclear transfer. Thus, in vitro-produced pluripotent cells can serve as donors of nuclei for cloning mice, but may be prone to chromosomal aberrations leading to a high rate of cloned embryo death.
49 TRANSCRIPT LEVEL OF mRNA IN BOVINE CLONED OR RE-CLONED EMBRYOS
Authors: Jang G. , Jeon H. Y. , Ko K. H. , Oh H. J. , Park H. J. , Hong S. G. , Kim J. J. , Kang S. K. , Lee B. C.
Published: December 2006

Gene expression in embryos played important roles during preimplantation development and had the potential to be used as an indicator for embryo viability. The purpose of this study was (1) to compare the developmental competence of cloned, or re-cloned embryos (Experiment 1); (2) to analyze the transcripts level of the related implantation, metabolic, and imprinting genes in IVF, cloned and re-cloned embryos (Experiment 2). The SCNT was performed according to the established system in our laboratory (Theriogenology 2006 65, 1800–1812). For producing cloned embryos, fetal fibroblasts as donor cells were used and a viable cloned calf was born. Recloned embryos derived from ear fibroblasts of the cloned calf, genetically same with donor fetal donor cells, were produced. The couplets were fused, chemically activated, and cultured in modified synthetic oviduct fluid (mSOF) for up to 7 days. The developmental competence up to blastocysts was observed under a microscope. The implantation (Bax, E-cad, If-tau, Hsp 70, Igf2r, and DNMT1), metabolic [LDHA (Lactate Dehydrogenase A), G6PD (Glucose-6 Phosphate Dehydrogenase), PGK (Phosphogycerate Kinase), Na/K ATPase, and Glut-1], and imprinting [GNAS (guanine nucleotide binding protein, alpha stimulating), UBE3a (ubiquitin protein ligase E3A), Mest (mesoderm specific transcript), SNRPN (small nuclear ribonucleoprotein polypeptide N), and Ndn (necdin)] genes were selected. The relative abundance (ratio to GAPDH mRNA) of gene transcripts in blastocysts was measured by conventional semi-quantitive RT-PCR. In Experiment 1, development competence of SCNT pre-implantation embryo was not different between cloned or re-cloned embryos (26% vs. 22%). In Experiment 2, the relative expression of Bax, Hsp70, If-tau, and Igf2r transcript was not different in IVF, cloned, and re-cloned embryos. Expression of E-cad and DNMT1 was higher in re-cloned embryos than any other group. Transcripts levels of LDHA, Na/K ATPase, and Glut-1 showed the similar relative abundance in IVF, cloned and re-cloned embryos. Expression of G6PD and PGF was increased in re-cloned and cloned embryos, respectively. Compared to IVF and cloned embryos, in re-cloned embryos, relative abundance of related imprinting genes (Ube3a, Mest, SNRPN, and Ndn) was increased. In conclusion, this study demonstrated that, whereas developmental competence of cloned or re-cloned embryos was not different, gene transcript levels were observed differently. It was suggested that alteration of gene expression in the re-cloned embryos derived from the cloned calf, genetically same with initial donor cells, might have affected the fetal development and births of re-cloned offspring.
51 PROTEOMICS ANALYSIS OF PLACENTOMEGALY IN CLONED MICE
Authors: Kim H. R. , Naruse K. , Lee H. R. , Wakayama T. , Park C. S. , Jin D. I.
Published: December 2006

A variety of mammalian species have been cloned during the past few years. However, the success rate of somatic cell nuclear transfer in animals has been extremely low with many problems. Particularly, placentomegaly is a frequent finding in cloned mice and cattle (Wakayma et al. 1999 PNAS USA 96, 14 984–14 989; Niemann et al. 2000 Theriogenology 53, 21–34). To assess protein expression profile in the placentomagaly of cloned mice produced by nuclear transfer of embryonic stem cells, we have used global proteomics approach by 2-D gel electrophoresis and mass-spectrometry with the differential protein patterns using the 3 placentae of cloned mice and 4 normal mouse placentae. Proteins within isoelectric point range pH 4.0~9.0 and molecular weight range of 20–100 kDa separately were analyzed by 2-D gel electrophoresis with 3 replications of each sample. A total of approximately 3500 spots were detected 2-D gel. In the comparison of normal and cloned placenta samples, a total of 49 protein spots were expressed differentially, of which 28 spots were up-regulated proteins including alpha-fetoprotein, aspartyl aminopeptidase, placental lactogen 2, tissue inhibitor of metalloproteinase 2 (TIMP-2), etc., and 21 spots were down-regulated proteins including peroxiredoxin 6, creatine kinase, pre-B-cell colony-enhancing factor 1 (PBEF), etc. Eight spots could not be identified. One of differentially up-regulated proteins in cloned mouse placenta was identified as TIMP-2 protein that is related to extracellular matrix degradation and tissue remodeling processes. Western blot was performed with placental sample used in the 2-D gel electrophoresis analysis and normal mouse placenta samples on Days 11.5 to 18.5. Indeed, Western blot analysis confirmed a significant increase of TIMP-2 protein level in cloned mouse placenta compared with normal. The expression levels of TIMP-2 in normal mouse placenta were highest at the normal mid gestation (Day 13.5) and exhibited prominent decrease at late gestation period during normal pregnancy. However, the expression levels of TIMP-2 in placenta of cloned mice appeared to be similar to levels of mid gestation normal mouse placenta. And one of down-regulated protein in NT placenta was identified as PBEF protein that is known to be related to induction of spontaneous labor. PBEF protein level in cloned mice placenta was revealed to decrease remarkably compared with normal. The expression levels of PBEF in normal mice placenta exhibited a gradual decrease between Day 11.5 and Day 18.5 without complete depletion. However, the expression levels of PBEF in placenta of cloned mice appeared to be markedly lower than those of the same day normal placenta. In conclusion, abnormal expression of placental proteins associated with tissue remodeling and labor induction may be the cases of placentomegaly in cloned mice.
52 EFFECT OF DONOR CELLS WITH VARIOUS DIFFERENTIATED STATUS ON THE DEVELOPMENTAL COMPETENCE OF PORCINE NUCLEAR TRANSFER EMBRYOS
Authors: Kim J. G. , Kang E. J. , Kim M. K. , Choe S. Y. , Rho G. J.
Published: December 2006

Adult stem cells are more desirable than somatic cells for nuclear transfer (NT) because of their easy reprogrammability to resemble the genome of the zygote (Zhu et al. 2004 Biol. Reprod. 70, 1088–1095). Mesenchymal stem cells (MSCs) are a heterogeneous population of uncommitted and lineage-committed cells and have a more flexible potential as donor cells for NT. The aim of this study was to compare the developmental potential of NT embryos using undifferentiated (MSCs) and differentiated cells in the same lineage (osteocyte, adipocyte, and chondrocyte) by assessing the cleavage and blastocyst rates. Fetal fibroblasts were used as NT control. MSCs obtained from the aspirated bone marrow of a neonatal pig were cultured in advanced-DMEM (ADMEM) supplemented with 5% FCS. The differentiation potential was demonstrated by culture of MSCs at passage 3 under the conditions that were favorable for adipogenic, osteogenic, and chondrogenic development (Pittenger et al. 1999 Science 284, 143–147). For NT, cells from passages 3–5 were transferred into the perivitelline space of enucleated MII oocytes that had been in vitro-matured after collection from slaughterhouse-derived ovaries. After fusion with a needle-type electrode, eggs were cultured in 7.5 µg mL−1 cytochalasin B for 3 h, and subsequently cultured in PZM-3 medium for 6 days. Statistical significance was tested using ANOVA with Bonferroni and Duncan tests. The results are presented in Table 1. The rates of cleavage and development to blastocyst stage of NT embryos varied among donor cell sources. Most eggs (92.2 ± 2.7%) cloned with MSCs cleaved, and 47.8% of eggs developed to the blastocyst stage. In contrast, NT eggs using differentiated MSCs—osteocytes, adipocytes, chondrocytes, and controls (fetal fibroblasts)—revealed significantly (P < 0.05) lower cleavage (74.5, 63.4, 74.3, and 66.4%, respectively) and blastocyst development (33.7, 30.1, 36.5, and 25.5%, respectively) rates than those using undifferentiated MSCs. The results demonstrate that the genome of donor cells with different differentiated status supports embryonic development to various degrees, and multipotent MSCs might have a greater potential in producing viable cloned porcine embryos.

Table 1.Development of NT embryos with undifferentiated and differentiated cells

This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.
53 EFFECTS OF INSULIN-TRANSFERRIN-SELENIUM IN DEFINED AND PORCINE FOLLICULAR FLUID-SUPPLEMENTED MEDIUM ON IN VITRO PRODUCTION OF PORCINE SCNT EMBRYOS
Authors: Kim Y. U. , Bhandari D. P. , Hossein M. S. , Park S. M. , Lee E. , Park S. W. , Jeong Y. I. , Lee J. Y. , Kim J. H. , Jeong Y. W. , Kim S. , Hwang W. S.
Published: December 2006

Insulin promotes the uptake of glucose and amino acids, and is beneficial for maturation of oocytes in vitro. Transferrin is an iron-transport protein and selenium is an essential trace element. Insulin-transferrin-selenium (ITS) together has been used in some in vitro maturation systems. The present study was designed to evaluate the effects of ITS in defined and porcine folicular fluid (pFF)-supplemented IVM medium on the glutathione (GSH) concentration, and on developmental competence after somatic cell nuclear transfer. ITS liquid media supplement (I-3146) was purchased from Sigma-Aldrich (St Louis, MO, USA). Basic IVM medium was TCM-199 supplemented with 10 ng mL-1 epidermal growth factor, 4 IU mL-1 pregnant mare serum gonadotropin (PMSG) and hCG and either 1% PVA (defined medium) or 10% pFF. Ten �g mL-1 insulin, 5.5 �g mL-1 transferrin, and 5 �g mL-1 selenium was used for the entire 44-h culture period. The GSH content of a gruop of 10 to 20 oocytes was determined by the dithionitrobezoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. Fetal fibroblasts were used as somatic cell donors and reconstructed embryos were cultured in mNCSU-23 medium for 168 h. Cleavage and blastocyst formation was observed at 48 h and 168 h, respectively. The quality of blastocysts was assessed by differential staining of the inner cell mass (ICM) and the trophectoderm (TE) cells. Each experiment was replicated for 5 times. The data were analyzed by one-way ANOVA, and Tukey was used as a posthoc test. The level of GSH production significantly varied in different culture conditions. The highest GSH concentration was observed in the pFF + ITS group (8.2 picomol/oocyte). A total of 116, 125, 126, and 120 reconstructed oocytes were cultured, and 10.1, 15.3, 17.2, and 21.8% blastocysts were observed for PVA, PVA + ITS, pFF, and pFF + ITS groups, respectively (P < 0.05). The numbers of inner cell mass, trophrectoderm cells, and total cells were significantly higher in the pFF + ITS group compared with the other groups. The average number of total cells in blastocysts was 31.9 � 1.8, 43.1 � 3.5, 46.7 � 4.9, and 52.3 � 6.7 for PVA, PVA + ITS, pFF, and pFF + ITS groups, respectively (P < 0.05). ITS supplement improved the developmental competence in both the defined and the pFF supplemented groups. We recommend supplementing porcine IVM medium with 10 �g mL-1 insulin, 5.5 �g mL-1 transferrin, and 5 �g mL-1 selenium.
54 SUCCESS OF MOUSE CLONING FROM AN OUTBRED STRAIN BY TRICHOSTATIN A TREATMENT AFTER SOMATIC NUCLEAR TRANSFER
Authors: Kishigami S. , Bui H. , Wakayama S. , Van Thuan N. , Hikichi T. , Mizutani E. , Ohta H. , Suetsugu R. , Wakayama T.
Published: December 2006

Although the somatic cloning technique has been used for numerous applications and basic research of reprogramming in various species, the extremely low success rates have plagued this technique for a decade. Further, in mice, the clonable strains have been limited mainly to the hybrid F1 strains such as B6D2F1. Recently, we have reported a new efficient cloning technique using trichostatin A (TSA) where reconstructed oocytes are activated by 5 mM strontium with 5 nM TSA for 6 h, followed by 3 h of culture in KSOM medium containing the same concentration of TSA. After the TSA treatment, cloned embryos were cultured in KSOM medium without TSA. This TSA treatment leads to a 2–5-fold increase in success rates for mouse cloning of B6D2F1 cumulus cells. In this study, to further test the validity of this TSA cloning technique, we tried to clone the adult ICR mouse, an outbred strain, which has never been directly cloned before. Only when TSA was used did we obtain both male and female cloned mice from cumulus and fibroblast cells of adult ICR mice with 4–5% success rates, which is comparable to 6–7% of B6D2F1. Thus, the TSA cloning technique now allows us to successfully clone outbred mice, demonstrating that this technique not only improves the success rates of cloning from the hybrid strains but also enables mouse cloning from normally unclonable strains. Further, our results provide insight into the mechanism underlying why only limited strains can be cloned using the current standard cloning technique.
55 DIFFERENTIAL TRANSCRIPTIONAL GENE EXPRESSION BETWEEN MINIATURE AND LANDRACE PIG FETUSES: IMPLICATION FOR PRODUCTION OF CLONED MINIATURE PIGS USING LANDRACE PIGS AS OOCYTE DONORS AND SURROGATE MOTHERS
Authors: Koo O. J. , Lee S. H. , Hossein M. S. , Kang S. K. , Kim D. Y. , Lee B. C.
Published: December 2006

Miniature pigs are regarded as a better organ donor breed for xenotransplantation because of their compatible organ size with human than any other pig breeds. The present study was performed to evaluate a somatic cell nuclear transfer (SCNT) system for producing cloned miniature pigs using Landrace pigs as oocyte donors and surrogate mothers. In Experiment 1, differential mRNA expression patterns of Day 30 gestation fetuses between miniature and Landrace breeds were compared using 13 610 cDNA microarray (based on Pig Genome Oligo sets; Qiagen, Valencia, CA, USA). In each breed, total mRNA from 3 fetuses was pooled before hybridization to minimize individual sample effect. With the fold-change test, 1551 cDNAs (11.40% of total) showed more than a 2-fold difference of intensity between the 2 breeds. In miniature fetuses, 252 genes were up-regulated and 1299 were down-regulated compared to Landrace ones. Among them some crucial genes related to implantation, including vascular endothelial growth factor (VEGF), vitronectin, and c-kit, were significantly down-regulated in miniature pig fetuses. In Experiment 2, in vitro developmental competence of SCNT embryos using fibroblasts from both breeds as nuclei donors were evaluated. In total, 352 miniature and 345 Landrace cloned embryos were cultured in vitro. There was no significant difference in fusion rate (78.78 vs. 77.48%), cleavage rate (69.8 vs. 65.3%), blastocyst rate (15.5 vs. 16.7%), and total cell number of blastocysts (48.0 ± 11.2 vs. 51.9 ± 17.5; all respectively). In Experiment 3, in vivo development was also monitored. In total, 1684 and 1354 SCNT embryos derived from miniature and Landrace pigs were transferred to 9 and 7 Landrace pig surrogate mothers, respectively. Overall, miniature embryos showed less in vivo developmental potency than Landrace ones; pregnancy rate at Day 30 of gestation (44 vs. 86%) and birth rate (11 vs. 43%) were low in miniature pig (based on the number of surrogates). Mean efficiency of SCNT embryo to term (0.24 vs. 1.55%) and mean litter size (4 vs. 7) were also low in miniature pigs. These results suggest that although in vitro development of SCNT embryos using recipient oocytes from Landrace pigs was similar between the 2 breeds, miniature pig embryos cannot interact with Landrace pig's reproductive tract properly and fail to implant, thus inhibiting fetal growth. In conclusion, cloned miniature pigs can be successfully produced using Landrace pigs as oocyte donors and surrogate mothers; however, the efficiency was very low due to transcriptional differences of fetuses between the 2 breeds.
The authors are grateful for a graduate fellowship provided by the Ministry of Education, through the BK21 program.
57 NO BENEFICIAL EFFECT OF AGGREGATION ON INTERFERON TAU ACTIVITY IN BOVINE CLONE OUTGROWTHS
Authors: Kurosaka S. , Ealy A. D. , McLaughlin K. J.
Published: December 2006

Extraembryonic failure is associated with the early developmental failure of bovine somatic cell clones. A pregnancy recognition signal, interferon tau (IFNτ), is a potential marker to qualify trophectoderm cells in ungulates. Aggregation of clone embryos is one of the methods to improve cloning efficiency in mouse (Boiani et al. 2003 EMBO J. 22, 5304–5312). Here, we evaluated the outcome of aggregating bovine clones using the bioactivity of IFNτ produced by clone blastocyst outgrowths. Clone embryos were produced from fibroblasts and cultured in vitro in SOF supplemented with fetal bovine serum at 39�C in an atmosphere of 5% CO2, 5% O2, and 90% N2. Aggregation of embryos was performed at the 8–16-cell stage by removing the zonae pellucidae with 0.5% pronase and placing 2 zona-free embryos into a microwell on the bottom of a culture dish. Embryos were cultured until Days 7–8. For outgrowth culture, blastocysts at Days 7–8 were individually placed on 4-well or 24-well plates containing mitotically inactivated STO feeder layers, and cultured at 37�C under 5% CO2 in air in 1 mL of DMEM with 10% FBS in each well. The medium from each well (1 mL) was collected for analysis and replaced at Day 7, 14, and then every 3 days until 50 days. IFNτ antiviral assay was performed as described previously (Talbot et al. 2000 Biol. Reprod. 62, 235–247). IFNτ activity of outgrowths derived from clone aggregates was compared with those from single clones and IVF embryos. The number of IFNτ-positive outgrowths from single and aggregate IVF embryos decreased gradually until Day 32 and Day 35, respectively. In contrast, positive outgrowths from both single and aggregate clone embryos decreased dramatically before Day 26. While all of the positive IVF outgrowths at Day 35 were still positive at Day 50, the number of positive clone outgrowths decreased after Day 35. At Day 50, 66.7% (4 of 6), 42.9% (3 of 7), 11.8% (2 of 17), and 0% (0 of 18) of outgrowths were positive for IFNτ activity in IVF singles, IVF aggregates, clone singles, and clone aggregates, respectively, consistent with the typical loss of somatic cell clone fetuses during the first trimester and development rates to term. We conclude that IFNτ bioactivity adequately reflected the in vivo development of clones vs. IVF embryos, but that clone–clone aggregation had no quantitative benefit on extended in vitro culture.
63 PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES RECONSTRUCTED BY CENTRIFUGATION AND ELECTROFUSION AFTER METAPHASE-II CHROMOSOME TRANSFER
Authors: Maedomari N. , Kikuchi K. , Fahrudin M. , Nakai M. , Ozawa M. , Noguchi J. , Kaneko K. , Nagai T. , Kashiwazaki N.
Published: December 2006

Metaphase-II chromosome transfer (M-II transfer) is considered to be a useful technique for studying nucleus–cytoplasm relationships, or for generating oocytes with good developmental ability after transfer of the nucleus to the cytoplasm. The reconstructed oocytes carry the original genomic information within the metaphase chromosomes from the donor oocytes. The objective of the present study was to evaluate the parthenogenetic developmental ability of porcine M-II transferred oocytes. In vitro maturation was carried out as reported previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). After culture for 44 h, cumulus cells were removed by hyaluronidase treatment and gentle pipetting. Oocytes that had extruded the first polar body were selected and centrifuged at 13 000g for 9 min to stratify the cytoplasm. The zonae pellucidae were removed after exposure to pronase, and zona-free oocytes were layered on a 300 �L discontinuous gradient (100 �L each of 45%, 30%, and 7.5%) of Percoll in TCM-HEPES supplemented with 5 �g mL-1 cytochalasin B. After centrifugation of the oocytes on the gradient in microcentrifuge tubes at 6000g for 20 s, fragmented cytoplasm with an equal volume was obtained, stained with Hoechst 33342, and classified as cytoplasm with or without chromosomes by observation with a fluorescence microscope. One fragmented cytoplasm with chromosomes and 2 fragmented cytoplasms without chromosomes were fused by electric stimulation with a single DC pulse (1.5 kV cm-1, 20 �s) and cultured temporarily for 1 h. The reconstructed oocytes were then stimulated again to induce parthenogenetic activation (0.8 kV cm-1, 30 �s, 2 DC pulses) (treatment group). Zona-free mature oocytes that had not been subjected to reconstruction were activated as a control group. The oocytes in both groups were treated with 5 �g mL-1 cytochalasin B for 2 h, and then cultured for 6 days in media (Kikuchi et al. 2002) using the WOW system (Gabor et al. 2000 Mol. Reprod. Dev.). The blastocyst formation rate in the control group (22.9 � 5.5%) was significantly higher (P < 0.05; ANOVA and PLSD-test) than that in the treatment group (7.6 � 1.8%). The total cell number per blastocyst in the control group (28.7 � 4.6) was significantly higher (P < 0.05) than that in the treatment group (16.7 � 1.0). These results suggest that reconstructed porcine oocytes following M-II transfer by centrifugation and electrofusion can develop to the blastocyst stage in vitro. This technique enables transfer of the nucleus to cytoplasm with good developmental ability without the use of a micro-manipulation system.
7 EFFECTS OF DIFFERENT TRYPSIN SOURCES AND CONCENTRATIONS ON THE VIABILITY OF BOVINE SPERM PRE- AND POST-CRYOPRESERVATION
Authors: Blevins B. A. , Steenson S. , Loskutoff N. M.
Published: December 2006

The goal of this research was to investigate the effect of different sources and concentrations of trypsin on the viability of bovine sperm as a potential method for removing pathogens similar to the washing methods developed for embryos. Trypsin derived from porcine pancreas (Sigma-Aldrich, St Louis, MO, USA) at 2.5% and 0.25% was compared to the recombinant human sequence (TrypLE Select; Invitrogen, Carlsbad, CA, USA) at 10× and 1× concentrations. Cryopreserved bovine sperm (n = 3 bulls) were thawed and processed using discontinuous (90/45) Percoll density gradient centrifugation; the sperm pellets were then washed (10 min at 300g) in TL-HEPES Solution (Cambrex Corp., East Rutherford, NJ, USA) and then resuspended in 1 mL of the same medium. Aliquots of 200 µL of the washed sperm were then added to 1 mL of each of the 4 trypsin treatments as well as a negative control (without trypsin) and incubated at room temperature. Aliquots (25 µL) of each treatment were examined for progressive motility after 5 min. As a result, the control sperm (no trypsin) increased progressive motility by 6.7% and the 1× TrypLE treatment by 9.3%. However, the 10× TrypLE Select and the 10× and 1× porcine pancreas extracts decreased progressive motility by 8.3, 29.0, and 4.0%, respectively. The objective of the second experiment was to determine if the treatment of bovine semen with trypsin (1× TrypLE Select and 0.25% porcine pancreas extract) before or after cryopreservation would affect sperm quality as compared to cryopreservation without trypsin treatment. Raw semen (n = 6 bulls) was collected, evaluated, cryopreserved, and then thawed using a standard bovine method (Biladyl®; Minitube, Verona, WI, USA) without further treatment (control) or after treatment with one of two trypsin treatments (density gradient centrifugation with 1× TrypLE Select or 0.25% porcine pancreas extract in the 45% Percoll layer and a soybean trypsin in activator (Sigma) in the 90% layer) either before freezing (Treat–Freeze) or after thawing (Freeze–Treat). The results for the 6 individual bull samples were comparable and are presented as means (± SEM) compared to the cryopreserved control (no trypsin treatment). Using the Mann–Whitney Rank Sum Test, no differences (P > 0.05) were found in any of the parameters comparing the crypreserved controls (no trypsin treatment) and the 4 treatments: Freeze–Treat vs. Treat–Freeze using either the recombinant TrypLE Select (1×) or the porcine pancreas extract (0.25%). These results suggest that trypsin treatment, before or after cryopreservation, can be used safely on bovine sperm without affecting viability in vitro.

Table 1.Comparison of cryopreserved bovine semen without and with various treatments
75 INHIBITION OF NK CELL CYTOTOXICITY IN CLONED MINI-PIGS EXPRESSING HUMAN LEUKOCYTE ANTIGEN-G1 (HLA-G1) GENE
Authors: Park K. W. , Han G. S. , Choi K. M. , Hong S. P. , Yoo J. Y. , Kim E. J. , Kim S. H. , Kim T. S. , Park S. Y. , Jun K. S. , Heo K. N. , Jin D. I. , Park C. S. , Seol J. G.
Published: December 2006

Human natural killer (NK) cell-mediated response plays an important role in xenograft rejection. In the case of pig-to-human xenotransplantation, it has been suggested that NK cells are involved in delayed-type rejection, which is characterized by pig endothelial cell (pEC) activation, direct lysis, and secretion of proinflammatory cytokines. NK cell activation can be a direct barrier to the potential use of pig organs for human xenograft transplantation. Therefore, it is important to suppress the NK cell activity on pig-to-human xenografts. Expression of HLA-G1 (non-classical major histocompatibility complex class I molecules) inhibits the cytotoxic activity of NK cells and has been proposed as a potential solution to overcome NK cell-mediated xenogeneic cytotoxicity in pEC. In this study, we transfected the HLA-G1 gene into mini-pig fetal fibroblasts to produce 2 HLA-G1 clonal cell lines. These cell lines were used to produce cloned HLA-G1 transgenic mini-pigs by nuclear transfer (NT). The presence of the HLA-G1 gene in transgenic mini-pigs was confirmed by PCR. The expression of HLA-G1 was detected by flow cytometry-immunohistochemistry assay. Mini-pig fibroblasts derived from a 35-day-old cloned fetus also showed characteristics similar to those of HLA-G1 clonal cell lines. The expressed HLA-G1 significantly suppressed NK-mediated cell lysis, and the rate of NK 92MI cell cytotoxicity was reduced as compared to the control group (HLA-G1: 46.7 � 4.5%; control: 4.6 � 13.3%; P < 0.05). In conclusion, transgenic cloned mini-pigs expressing HLA-G1 were produced by NT for the first time. It is expected that these mini-pigs could be used to overcome the NK cell-mediated rejection in xenotransplantation.
81 MOUSE CLONING BY USING A LASER-ASSISTED ZONA OPENING AND ELECTRO-FUSION TECHNIQUE
Authors: Rungsiwiwut R. , Thongphakdee A. , Numchaisrika P. , Virutamasen P. , Techakumphu M.
Published: December 2006

Mouse cloning can be performed by a direct microinjection of donor nuclei using a conventional or a piezo-actuated technique (Rybouchkin et al. 2002 Reproduction 124, 197–207; Wakayama et al. 1998 Nature 394, 369–374). However, a high percentage of lysed oocytes was observed during the pipette penetration of the cytoplasmic membrane through the zona pellucida. The aim of this experiment was to investigate the possibility of a combination of a laser-assisted zona opening and electro-fusion for mouse cloning. Mature oocytes were obtained from FSH-superovulated B6D2F1 female mice. Enucleation and transfer of donor cell were performed in HEPES-buffered CZB medium. Spindle-chromosome complexes (SCCs) together with first polar body were removed by blunt-end pipette via a small hole in the zona pellucida which was cut by a laser beam. An adult fibroblast cell was introduced into the perivitelline space and fused to the enucleated oocyte by using a single DC pulse of 1.5 kV cm-1, 20 �s, in a fusion medium (Liu and Aoki 2003 Animal Sci. J. 75, 125–129). The fusion rate was checked 30 min later and only the fused oocytes were subjected to activation by 6 h culture in Ca2+-free CZB medium supplemented with 10 mM Sr2+ and 5 �g mL-1 cytochalasin B. The oocytes which presented the pseudo-pronuclei were considered as the activated oocytes and were cultured in CZB medium at 37�C, 5% CO2 in humidified atmosphere. The developmental rate was observed every 24 h for 4 days. The diploid parthenogenetically activated embryos serving as a control were obtained using the same activation protocol but without SCC removal. The percentages of survival after enucleation and after fusion were recorded. The formation of pseudo-pronuclei and the embryos developing to a particular stage were determined by chi-square analysis. The results show that most of the oocytes survived after enucleation (92.5%, 172/186) and the fusion rate was 71.9% (105/146). The formation of pseudo-pronuclei and the cleavage rate of cloned embryos was lower than in the control (87.6% (92/105) vs. 100% (90/90) and 69.6% (64/92) vs. 92.2% (83/90), respectively). The developmental rate to morula–blastocyst stage of cloned embryos was significantly lower than in the control [1.1% (1/92) vs. 44.4% (40/90); P < 0.05]. These results indicate that using laser-assisted zona opening and electro-fusion technique is practical for mouse cloning and provides an alternative method when injection of donor nuclei into the recipient oocytes using a conventional or a piezo-driven method is technically difficult.
This study was supported by grants from The National Research Council of Thailand and The Thailand Research Fund (Loyal Golden Julilee Ph.D. program).