bluuebunny/crossref_metadata_2025 huggingface dataset:
Photoluminescence and polarized photodetection of single ZnO nanowires
Authors: Fan Zhiyong , Chang Pai-chun , Lu Jia G. , Walter Erich C. , Penner Reginald M. , Lin Chien-hung , Lee Henry P.
Published: December 2004

Single crystal ZnO nanowires are synthesized and configured as field-effect transistors. Photoluminescence and photoconductivity measurements show defect-related deep electronic states giving rise to green-red emission and absorption. Photocurrent temporal response shows that current decay time is significantly prolonged in vacuum due to a slower oxygen chemisorption process. The photoconductivity of ZnO nanowires is strongly polarization dependent. Collectively, these results demonstrate that ZnO nanowire is a remarkable optoelectronic material for nanoscale device applications.
Room temperature ferromagnetism in anatase Ti0.95Cr0.05O2 thin films: Clusters or not?
Authors: Hong Nguyen Hoa , Ruyter Antoine , Prellier W. , Sakai Joe
Published: December 2004

Laser ablated Cr-doped TiO2 thin films grown on LaAlO3 substrates are single phased anatase and room temperature ferromagnetic. The magnetic moment of Cr-doped TiO2 films is rather large, and it is consistent with the theoretical predictions. Magnetic force microscopy measurements certainly suggested that the strong ferromagnetism at high temperature in Cr-doped TiO2 films is intrinsic, and it must originate from the diluted magnetic matrix but not from any form of clusters.
Nanodispenser for attoliter volume deposition using atomic force microscopy probes modified by focused-ion-beam milling
Authors: Meister A. , Liley M. , Brugger J. , Pugin R. , Heinzelmann H.
Published: December 2004

In this letter, we describe the on-demand dispensing of single liquid droplets with volumes down to a few attoliters and submicrometric spacing. This dispensing is achieved using a standard atomic force microscope probe, with a 200nm aperture at the tip apex, opened by focused ion beam milling. The inside of the tip is used as reservoir for the liquid. This maskless dispensing, realized in ambient environment, permits the direct creation of droplet arrays. Nanoparticles, suspended in the liquid, were organized on a surface.
In
-rich In1−xGaxN films by metalorganic vapor phase epitaxy
Authors: Chang Chin-An , Shih Chuan-Feng , Chen Nai-Chuan , Lin T. Y. , Liu Kuo-Shiun
Published: December 2004

Single crystalline In1−xGaxN films containing high In content (70%–100%) were grown by metalorganic vapor phase epitaxy. A linear relation was observed between the lattice constants and gas phase Ga∕In ratios. The surface morphology changed from pyramid for InN to more planar ones for the InGaN alloys with increasing Ga content. The electron mobility decreased rapidly from 1200cm2∕Vs for InN to less than 100cm2∕Vs for In0.7Ga0.3N, with a carrier concentration of low- 1019cm−3 for all the as-grown films. Using photoluminescence a single emission peak was observed at 1.4–1.6μm for the In-rich InGaN with decreasing wavelengths up to below 20% of Ga. Two peaks were observed for the In0.80Ga0.20N, however, indicating possible phase separation. The x-ray photoelectron spectroscopic measurement showed shifts to higher binding energies for both In and Ga with increasing Ga content. The estimated alloy composition, however, depended sensitively on the sputtering conditions of the samples.
Role of the mitochondrial genome in preimplantation development and assisted reproductive technologies
Authors: Smith Lawrence C. , Thundathil Jacob , Filion France
Published: December 2004

Our fascination for mitochondria relates to their origin as symbiotic, semi-independent organisms on which we, as eukaryotic beings, rely nearly exclusively to produce energy for every cell function. Therefore, it is not surprising that these organelles play an essential role in many events during early development and in artificial reproductive technologies (ARTs) applied to humans and domestic animals. However, much needs to be learned about the interactions between the nucleus and the mitochondrial genome (mtDNA), particularly with respect to the control of transcription, replication and segregation during preimplantation. Nuclear-encoded factors that control transcription and replication are expressed during preimplantation development in mice and are followed by mtDNA transcription, but these result in no change in mtDNA copy number. However, in cattle, mtDNA copy number increases during blastocyst expansion and hatching. Nuclear genes influence the mtDNA segregation patterns in heteroplasmic animals. Because many ARTs markedly modify the mtDNA content in embryos, it is essential that their application is preceded by careful experimental scrutiny, using suitable animal models.
Comparison of neurosphere cells with cumulus cells after fusion with embryonic stem cells: reprogramming potential
Authors: Do Jeong Tae , Schöler Hans R.
Published: December 2004

Embryonic stem cells (ESCs) are the pluripotent cells that also have the capacity to induce the genomic reprogramming of differentiated somatic cells. The progressively restricted genomic potential of somatic cells observed during embryonic development can be reverted to a pluripotent state by the formation of cell hybrids with ESCs. To assess the reprogramming potential of ESCs, we investigated the reprogramming of one of two different somatic cell populations, neurosphere cells (NSCs) and cumulus cells (CCs), after fusion with ESCs. Specifically, hybrid cells were produced by cell fusion of E14 ESCs with either NSCs or CCs containing the neo/lacZ and Oct4-GFP transgenes. The first reprogramming event, observed by the presence of Oct4-GFP in the hybrid cells, could be identified on Day 2, at approximately 45 h after fusion in both ESC-NSC and ESC-CC hybrids. In addition, the two ESC–somatic cell hybrids exhibit a similar reprogramming rate and share characteristics with the E14 ESC line: (1) expression of pluripotent markers (Oct4, Rex-1 and nanog); (2) inactivation of differentiated tissue-specific gene expression; and (3) the capacity to differentiate into all three germ layers. Taken together, our results suggest that the ESC–somatic cell hybrids have fully acquired ESC characteristics and that somatic cells of different tissue origin have the same potential to be reprogrammed after fusion with ESCs.
137 EFFECT OF FLUNIXIN MEGLUMINE IN CO-CULTURE MEDIUM ON THE DEVELOPMENT OF IN VITRO MATURED AND FERTILIZED BOVINE EMBRYOS
Authors: Goda S. , Hamano S. , Miyamura M. , Dochi O. , Koyama H.
Published: December 2004

PG concentration is often increased during uterine manipulation with embryo transfer. Embryo viability is affected by the increase in the PGF2α concentration accompanying manipulation of the uterus during embryo transfer. Schrick et al. (2001 Theriogenology 55, 370 abst) observed that treatment with flunixin meglumine, an inhibitor of prostaglandin, increased pregnancy rates depending on the stage and quality of embryos transferred. On the other hand, prostaglandin was secreted by a cumulus cell monolayer in an in vitro culture of bovine oocytes. The present study aimed to assess the effects of flunixin meglumine in culture medium on the development of in vitro-matured and fertilized bovine embryos. COCs were collected from ovaries of slaughtered cows by aspiration. The COCs were matured for 20 h in TCM-199 supplemented with 5% fetal bovine serum (FBS) and antibiotics at 38.5°C under an atmosphere of 2% CO2 in air. Matured COCs were inseminated with 1.0 × 107 sperm mL−1 in BO medium (Brackett and Oliphant 1975 Biol. Reprod. 12, 260–274) containing 5 mM theophillin and 5 μg mL−1 heparin for 5 h. All of the inseminated oocytes were introduced into the maturation medium that had been kept with the cumulus cells in the CO2 incubator. At 48 h after insemination, all embryos over the 4-cell stage were cultured in TCM-199 plus 5% FBS supplemented with each of five concentrations of flunixin meglumine (0, 0.0025, 0.005, 0.01, and 0.025%) with a cumulus cell monolayer. Development to the blastocyst stage and quality were examined at Days 7 to 8 (Day 0 = day of insemination) using a microscope. The experiment was replicated four times. Data were analyzed by the chi-square test. The total blastocyst rates from the over-4-cell embryos were 61.2 (52/89), 53.7 (44/89), 65.6 (59/90), 57.3 (51/89), and 33.7% (31/92) for 0, 0.0025, 0.005, 0.01, and 0.025%, flunixin meglumine, respectively. The total blastocyst rate with the flunixin meglumine concentration of 0.025% was significantly lower than those with the other concentrations (P < 0.05). The proportion of grade 1 blastocysts with the flunixin meglumine concentration of 0.005% was significantly higher than that with the 0, 0.0025, and 0.025% concentrations (27.8 vs 11.2, 14.6, and 5.4%; P < 0.05). Our present results show that the addition of 0.005% flunixin meglumine to the co-culture medium is positively associated with blastocyst quality in bovine embryos.
190 A PROCEDURE TO OBTAIN FIBROBLASTS FROM WILD ANIMALS
Authors: Caamaño J.N. , Hermsen M. , Marcos J. , Meana A. , Alonso C. , Goyache F. , Prieto M. , Espí A. , Royo L.J. , Diez C. , Pajares G. , Villanúa D. , Borragán S. , Prather R.S. , Gómez E.
Published: December 2004

The number of wild animals threatened or at risk of extinction constantly increases and, as a result, biodiversity decreases. Therefore, it is necessary to set in motion proceedings to preserve the genetic variability and to overcome factors leading to this situation. Banks of cryopreserved cells, such as skin fibroblasts, from wild animals could be used both for genetic studies and as a cell source for technologies able to reproduce individuals. In this study we report that a single basic protocol provides a method for obtaining, culturing, and cryopreserving skin fibroblasts from a wide range of wild animals. Skin biopsies were taken in Cantabria (Spain) from live, anesthetized brown bear (Ursus arctos, n = 4), and in Asturias (Spain) from carcasses of accidentally killed individuals from the following species: grey wolf (Canis lupus, n = 1), red fox (Vulpes vulpes, n = 1), eurasian badger (Meles meles, n = 1), ermine (Mustela erminea, n = 1), fallow deer (Dama dama, n = 7), pyrenean chamois (Rupicapra pyrenaica, n = 1), western roe deer (Capreolus capreolus, n = 1), wild boar (Sus scrofa, n = 1), striped dolphin (Stenella coeruleoalba, n = 1) and one avian species, capercaillie (Tetrao urogallus, n = 1). Once obtained and transported to the laboratory, all samples were processed identically. Tissue samples were manually cut into small pieces and enzymatically digested with 0.05% trypsin-EDTA (Sigma-Aldrich, Madrid, Spain) for 30 min, and then with collagenase (300 units/mL) (Sigma) for 14 h at 38°C. Disaggregated cells were centrifuged at 1600 rpm for 10 min and the pellet was diluted with D-MEM (Sigma) containing 10% FBS. Cells were counted and a minimum of 3 × 105 cells were placed in a 25 cm2 flask for culture under 5% CO2 in air and high humidity at 37°C (red fox, badger, ermine, western roe deer) or 38°C (brown bear, grey wolf, fallow deer, pyrenean chamois, wild boar, striped dolphin, capercaillie). Confluent fibroblast monolayers were obtained after 5–7 days in culture except for the striped dolphin whose sample was contaminated. Two to three passages were performed using 75 cm2 flasks before freezing fibroblasts in D-MEM containing 10% DMSO (Sigma) and 10% FBS. Total cell yield from a confluent flask was 5–11 × 106 cells, depending on the animal species. Fibroblasts recovered from all thawed samples grew to confluence, and karyotypes were performed at this step. It was possible to obtain, culture, freeze, and thaw skin fibroblasts successfully from all the above-mentioned land species. Although more research is needed, preliminary data suggest that the above-described procedure can be applied to mammals and birds, thus helping to preserve genetic material of wild animals.
Grant Support: Consejería de Medio Ambiente, Ordenación del Territorios e Infraestructuras, Asturias, SPAIN. The authors acknowledge CEPESMA, Luarca, Asturias, SPAIN.
203 ASSESSMENT OF OVARIAN RESERVE. IS THERE A ROLE FOR OVARIAN BIOPSY?
Authors: De Roover R. , Hanzen C.
Published: December 2004

The pool of primordial follicles in the ovary or ovarian reserve is a major factor in human fertility potential. In bovine medicine as well, this ovarian reserve has been linked to the results of superovulation procedures (Cushman et al. 1999 Biol. Reprod. 60, 349–354). These authors suggested a biopsy to assess the level of this reserve. Whether the biopsy(ies) is(are) a true reflection of the follicular distribution in the ovarian cortex, is (to the best of our knowledge) a factor never investigated until now in bovine medicine. In human medicine, this procedure has been critically examined for that particular use and found not to be suited (Lass et al. 2004 Hum. Reprod. 19, 467–469). Indeed, randomized or “blind” sampling of one biopsy is adequate only if follicles are evenly spread in the ovarian cortex; in any case they are not deeper than a few mm from the surface. Moreover, the quantitative counting of follicles does not provide any information about the quality of the oocytes embedded in them. Taking a biopsy of a bovine ovary in a minimally invasive way is technically feasible (Aerts 2004 Reprod. Fertil. Dev. 16, 229–230). Therefore, the aim of this study was to examine the natural distribution of primordial follicles in the ovarian cortex of bovine ovaries. Slaugtherhouse ovaries were collected at random. The volume (mL) was measured and the macroscopically visible follicles were counted. Then the ovaries were cut in slices of 5Âμm, and every 8th (8 × 5 = 40 μm interval) slice was subjected to fixation in formalin and hematoxylin-eosin staining. Before counting of the primordial follicles, the ovarian cortex was subdivided into 8 equal parts. These “parts” were supposed to mimick a (single) ovarian biopsy. The 8 parts of a slice represent here multiple biopsies. For each of these parts, the number of primordial follicles was counted; only follicles with a visible oocyte were included. The results of the parts containing the ligament of the ovary were excluded. Results are shown in Table 1. The results show that the distribution of primordial follicles between small parts of the bovine ovarian tissue was extremely uneven. A large variation was observed between samples obtained from the same ovary. Moreover, an extrapolation of follicle numbers found in biopsies to entire ovaries were hampered by the uneven size and morphology of these ovaries. Therefore, we conclude that the use of single biopsies of ovarian cortex for a quantitative evaluation of the ovarian reserve has limited value; an empty cortex or a cortex with very few follicles might be just incidental and meaningless. Even the use of multiple biopsies, although less variable, does not solve the problem of extrapolation of these data to entire ovaries.

Table 1.
Macroscopically visible follicles on 4 ovaries and primordial (“microscopical”) follicles on 4 slices of each of these ovaries
215 COMPARISON OF STRONG ENDOTHELIAL CELL-SPECIFIC PROMOTERS FOR EXPRESSION OF HUMAN COMPLEMENT REGULATORY PROTEINS IN PORCINE XENOGRAFT ENDOTHELIAL CELLS
Authors: Choi T.W. , Kim J.H. , Choi H.S. , Uhm S.J. , Park C.S. , Lee H.T. , Cho S.G.
Published: December 2004

The complement system is composed of a complex group of soluble proteins that have important roles in the immune response against foreign cells such as xenografted tissue cells. Activation of complement pathways results in the formation of the membrane attack complex (MAC). Several cell surface regulators known as membrane complement regulatory proteins (mCRPs) prevent the formation of MAC. For blocking the formation of MAC in the xenotransplanted cells, we tried to overexpress mCRPs in the endothelial cells to prevent the hyperacute rejection (HAR), because one of the major obstacles to xenotransplantation of vascularized organs was reported to be HAR. For this purpose, we isolated human MCP (membrane cofactor protein, CD46) (1.5 kb) and DAF (decay-accelerating factor, CD55) (1.1 kb) promoter regions from human genomic DNA isolated from the human embryonic kidney cell line HEK293, and cloned into pGL3 plasmid for luciferase reporter assay. We also inserted other known endothelial cell-specific promoters, such as Flk-1 (fetal liver kinase-1) and ICAM-2 (intercellular adhesion molecule-2), thrombomodulin promoters, into pGL3 vector to compare their promoter activities. We transfected the plasmids into several endothelial cell lines such as bovine aortic endothelial cells (BAEC), human umbilical vein endothelial cells (HUVEC), and mouse pancreatic microvascular endothelial cells (MS1). We also used two epithelial cell lines, human embryonic kidney epithelial cells (HEK293) and human epidermal keratinocytes (HaCaT). The endothelial specific expression of the promoters were compared with those of SV40, CMV, and EF-2a (elongation factor) promoters which are generally used in mammalian gene expression. Luciferase assays showed that among the endothelial cell-specific promoters, the 1.1 kb DAF promoter was the strongest, followed by the Flk-1 promoter. The promoter activity of 1.1 kb DAF was about 2-fold that of the Flk-1 promoter. Moreover, the 1.1 kb DAF and Flk-1 promoters were chosen as the best endothelial specific promoters, as both showed about 5 times more luciferase activity in the endothelial cells compared to that in the epithelial cell lines. We also found that the 5′-flanking region between −1126 and −968 of the 1.1 kb DAF promoter was very important for the endothelial-specific strong gene expression. On the 0.2 kb region of DAF promoter, we could detect several conserved nucleotide sequences interacting with the specific transcription factors, such as GATA-1, USF, CdxA, SRY, Sox-5, and Sp1. Interestingly, deletion of the GATA-1 motif between −1113 and −1104 reduced the promoter activity of the DAF promoter by about 25%. We conclude that the 1.1 kb DAF promoter is a suitable candidate promoter for strong endothelial cell-specific expression of mCRP genes.
22 INFLUENCE OF SIRE AND SIRE BREED (GIR VS. HOLSTEIN) ON EARLY PREGNANCY AND EARLY EMBRYONIC LOSS RATES IN HOLSTEIN COWS DURING SUMMER HEAT STRESS
Authors: Pegorer M.F. , Vasconcelos J.L.M. , Hansen P.J. , Barros C.M.
Published: December 2004

Heat stress has negative effects on pregnancy rates of lactating dairy cattle. There are genetic differences in tolerance to heat stress, and it has been shown that Bos indicus cattle are more resistant to high temperatures than Bos taurus. In the present work, effect of sire and sire breed on conception rate of Holstein cows during the Brazilian summer (January and February, 2003 and 2004) was determined. Lactating Holstein cows (n = 1113), producing an average of 25.2 kg milk/day and at 247 ± 136 days postpartum, were AI approximately 12 h after estrus detection (n = 433) or fixed-time AI (FTAI) after hormonal induction of ovulation (n = 680), using semen from 4 Gir (Bos indicus dairy bull, 657 AI) and 3 Holstein sires (Bos taurus, 456 AI). Pregnancy diagnosis was performed by ultrasonography or rectal palpation at 28–45 and 70–84 days after AI. Cows diagnosed pregnant at the first examination and open at the time of the second were considered as having lost their embryo. The results were analyzed by logistic regression using PROC GENMOD: the model considered the effect of sire, sire breed, inseminators, milk production 7 days before AI, period of lactation, and AI type (FTAI vs. AI after estrus detection). There was no significant effect of sire breed on early pregnancy rate (EPR: Gir = 78/657, 11.9%; and Holstein = 30/456, 6.6%, P = 0.34) or early pregnancy loss (EPL: Gir = 25/78, 32%; and Holstein = 10/30, 33.3%, P = 0.73). However, there was a sire effect on pregnancy loss (43.7, 35.3, 10, and 40% for Gir bulls 1, 2, 3, and 4, respectively) and (50, 18.2, and 38.5% for Holstein bulls 5, 6, and 7, respectively), with bull 3 having the lowest rate of pregnancy loss (P = 0.03). There was no effect of the AI type on EPR (FTAI = 65/680, 9.5%; and AI after estrus = 43/433, 9.9%; P = 0.8). Surprisingly, there were higher pregnancy losses in cows AI after estrus detection (17/43, 39.5%) as compared to FTAI cows (18/65, 27.7%; P = 0.03). The variables days at postpartum, milk production 7 days before AI, and inseminator did not significantly influence EPR or EPL. It is concluded that use of Bos indicus sires did not improve EPR of lactating Holstein cows during summer as compared to use of Bos taurus sires. However, some bulls, either indicus or taurus, were more effective in decreasing EPL and selection of bulls by this criterion may result in higher parturition rates in lactating Holstein cows.
This work was supported by USDA IFAFS Grant # 2001-52101-11318.
220 AN EXPRESSION PROFILE OF GENES CRUCIAL FOR PLACENTAL DEVELOPMENT IN SINGLE IN VIVO, IN VITRO AND CLONED BOVINE BLASTOCYSTS
Authors: Hall V. , Ruddock N. , Tecirlioglu R. , Cooney M. , French A.
Published: December 2004

Abnormalities of the placenta are a major factor contributing to early death in cloned bovine conceptuses. This is primarily due to incomplete chromatin remodeling and reprogramming of the donor nucleus. It is unknown whether genetic aberrations of genes crucial for placental development can be detected in pre-implantation cloned bovine embryos. This study looked at the expression profile of four genes in single bovine blastocysts derived from in vivo, in vitro produced (IVP), or cloning techniques, including handmade cloning (HMC) and serial HMC (SHMC). The genes studied included acrogranin, caudal type homeobox 2 (cdx2), estrogen-receptor-related receptor beta (essrb), and the mammalian relative of DnaJ (MRJ). These genes play a role in trophoblast regulation and placental development. Messenger RNA expression was analyzed by using PCR following cDNA amplification by means of SMART cDNA synthesis (Clontech, Palo alto, CA, USA). Primers were designed from homologous human and mouse sequence. PCR products were sequenced for verification. Five single blastocysts were analyzed from each of the following treatments: in vivo, IVP, HMC, and SHMC. Pooled (n = 10) IVP blastocyst cDNA produced by standard RT was used as a positive control. Grade 1 Day 7 blastocysts were selected for all treatments. Amplified cDNA was tested using control genes polyA, IFN-τ and GDF9. In vitro-produced embryos were matured, fertilized and cultured as published by Ruddock et al. (2004 Biol. Reprod. 70, 1131). Cloned HMC embryos were produced as described by Tecirlioglu et al. (2003 Reprod. Fertil. Dev. 15, 361). Serial HMC embryos were produced as per the HMC embryos, followed by a second round of nuclear transfer at the pronuclear stage. The pooled IVP, in vivo, and IVP blastocysts expressed all four genes of interest. In the HMC-cloned embryos, all four genes were expressed. However, in the SHMC cloned embryos, although MRJ was found to be expressed in all blastocysts, three of the five blastocysts did not express acrogranin. Similarly, two SHMC embryos did not express cdx2, and essrb was weakly expressed in three of the five embryos analyzed. Initial pregnancy rates of HMC and SHMC embryo transfers are similar. Further pregnancy results are pending. These results indicate that aberrations of genes crucial for placental development can be detected in single cloned blastocysts. It also suggests that failed implantation and/or placental defects may stem from patterned genetic defects in the pre-implantation embryo. An increase in the number of embryos analyzed would further strengthen results. These genes could act as markers to identify cloning techniques that produce more embryos with normal genetic profiles. The benefits of developing a screening tool to assess abnormalities in single pre-implantation embryos would be significant.
230 EFFECT OF IN VITRO CULTURE TREATMENT AND DONOR EWE DIET ON IGF2R EXPRESSION IN FETAL TISSUES
Authors: Powell K.A. , Mackie K. , McEvoy T.G. , Sinclair K.D. , Robinson J.J. , Ashworth C.J. , Young L.E. , Wilmut I. , Rooke J.A.
Published: December 2004

In separate experiments, elevation of donor ewe plasma urea levels and IVC of embryos in serum have been associated with occurrence of large offspring syndrome (LOS). Fetuses displaying LOS also have abnormal expression of the imprinted gene IGF2R. The present study, therefore, examined the effect of variation in donor ewe nitrogen metabolism and embryo culture on fetal development. Zygote-donor ewes were offered a diet with either a 0% (LN) or 3% urea supplement (HN) for two weeks during which superovulation and artificial insemination (AI) took place. Zygotes were recovered 36 h after AI and cultured for 5 days in synthetic oviductal fluid (SOF) to which either steer serum (10%; SOFS) or albumin (0.4%; SOFA) was added. In vivo control (C) blastocysts were recovered (Day 5) from superovulated ewes offered diet LN. All embryos were transferred singly to recipient ewes; fetal tissues were recovered and snap-frozen at Day 125 of gestation. IGF2R expression was measured by semi-quantitative PCR in fetal heart and kidney and expressed as the ratio to 18S mRNA. Data were analyzed by ANOVA with donor ewe plasma urea-N prior to (preUN) and mean plasma urea-N during (meanUN) feeding as covariates. Correlation analysis was used to test relationships between variables. Heart IGF2R expression (see Table 1) was lower in SOFA than in SOFS fetuses (P = 0.003; preUN, P = 0.007), while kidney IGF2R expression was lower in HN than in LN fetuses (P = 0.04; preUN, P = 0.015; meanUN, P = 0.032). Overall the range of both heart and kidney IGF2R expression was greater for fetuses from cultured (1st/3rd quartiles; heart, 0.87–1.05; kidney, 0.77–1.10) than from C embryos (heart, 1.03–1.16; kidney, 0.84–1.00). In IVC fetuses, heart IGF2R expression was negatively associated with fetal body (r = −0.31; P = 0.003) and heart weight (r = −0.35; P = 0.008) whereas kidney IGF2R expression was not associated (r = −0.23; P = 0.089) with fetal body weight and not related to kidney weight. Heart and kidney IGF2R expression were only weakly associated (r = 0.26; P = 0.054). In conclusion, changes in IGF2R expression in both fetal heart and kidney were associated with donor ewe N status and IVC conditions. In this study, decreases in heart IGF2R expression were reflected in increased heart weight, but kidney IGF2R expression was not related to kidney weight.

Table 1.
Fetal (kg), organ weights (g), and IGF2R expression

This study was funded by Defra.
244 EFFECT OF β-MERCAPTOETHANOL IN A TRANSIENT-IVF SYSTEM ON SPERM PENETRATION INTO PORCINE IVM OOCYTES
Authors: Funahashi H.
Published: December 2004

This study was carried out to determine the effect of β-mercaptoethanol (bME; at 50 μM) during a brief co-culture and the additional culture of inseminated oocytes on sperm penetration of porcine IVM oocytes. Cumulus-oocyte complexes (COCs) collected from 3–∼6-mm follicles were cultured in modified NCSU-37 supplemented with bME, eCG, hCG, and dibutyryl cAMP for 20 h and then in the same medium without eCG, hCG, and dibutyryl cAMP for additional 24 h. After IVM, the oocytes were denuded with 0.1% hyarulonidase, randomly divided into three groups (as described below) and fertilized in vitro. Treatment 1 (T1, control): Oocytes were co-cultured with fresh spermatozoa for 10 min in modified M199 medium (mM199) supplemented with 5 mM caffeine (+caff) in the absence of bME (bME-), then washed gently with caffeine-free mM199, and cultured for further 9 h in mM199; T2: co-culture for 10 min in mM199+caff bME− and additional culture in mM199 in the presence of bME (bME+); and T3: co-culture for 10 min in mM199+caff bME+ and additonal culture in mM199 bME+. At 9 h after insemination, oocytes were fixed and stained for the evaluation of sperm penetration. Results from four replicates were analyzed by ANOVA and Duncan's multiple range test. Sperm penetration rate was lower in T3 (79.8% in 92 oocytes) compared with T1 (95.3% in 84 oocytes) and T2 (93.3% in 93 oocytes). Percentage of eggs with male and female pronuclei in penetrated oocytes was higher in T1 (89.6%) than in T2 (56.4%) and T3 (57.2%). Monospermy rate was lower in T1 (48.9%) than in T2 (59.5%) and T3 (64.3%). These results demonstrated that, although the presence of β-mercaptoethanol during co-culture and the additional culture reduced the fertilization rate, the presence during the additional culture period affected the process of fertilization and increased the incidence of monospermy at 9 h after insemination. In conclusion, the progression of sperm penetration may be controlled by utilization of β-mercaptoethanol in porcine IVF system.
This work was supported by JSPS-KAKEN (C16580230).
246 FOLLICULAR DEVELOPMENT AND OOCYTE QUALITY AFTER OVUM PICKUP IN DONOR COWS
Authors: Imai K. , Tagawa M. , Matoba S. , Narita M. , Saito N.
Published: December 2004

The present study was designed to assess the renewal of follicular development and oocyte quality after ovum pickup (OPU) in Holstein dry cows. Cows were kept under the same feeding and environmental conditions. In Experiment 1, follicle aspiration (more than 2 mm) by OPU using a 7.5 MHz linear transducer with needle (cova needle, Misawa Medical, Tokyo, Japan) connected to an ultrasound scanner (SSD-1200, ALOKA, Tokyo, Japan) was performed in four cows. After OPU ovaries were observed from Day 4 (Day 0 = the day of OPU) to Day 11 to assess the number of follicles that developed. In Experiment 2, two sessions of OPU (n = 11) were performed with a 7-day interval between to assess the quality of developing follicles and oocytes. Oocytes were evaluated by their cumulus cell morphology, cytoplasmic color, and density. To assess the developmental competence of oocytes, collected cumulus-oocyte complexes (COCs) were cultured for 20 h in TCM-199 supplemented with 5% calf serum (CS) in a microdroplet (volume was adjusted to 5 μL/oocyte) at 38.5°C under atmosphere of 5% CO2 in air. After maturation, the COCs were inseminated with frozen-thawed semen collected from the same ejaculation of a single bull. The fertilization was performed with BO solution as described by Imai et al. (2002 J. Vet. Med. Sci. 64(10), 887–891). The putative zygotes were then cultured in CR1aa supplemented with 5% CS under the same conditions as maturation culture for nine days. Embryo development was assessed by the cleavage rate on Day 2 and the blastocyst formation rate on Days 7 to 9 (the day of insemination = Day 0). Data were analyzed by ANOVA or Student's t-test. In Experiment 1, the mean number of developing follicles (larger than 2 mm in diameter) were increased from Day 4 to Day 11 (Day 4: 19.8 ± 10.0, Day 7: 32.5 ± 9.5; Day 11: 39.5 ± 10.7 (mean ± SD), respectively, P < 0.05). In Experiment 2, the mean number of developing follicles and collected oocytes on the day of OPU were significantly (P < 0.05) different between the first and second sessions (54.2 ± 12.4 and 40.8 ± 12.7, 45.7 ± 20.2 and 27.7 ± 8.7, respectively). The percentage of Grade 1 and 2 oocytes for the first session was significantly lower (P < 0.05) than those for the second session (59.1 ± 8.4 and 69.0 ± 11.8), and no significant differences were found within cleavage and blastocyst rates. The mean numbers of blastocysts obtained per session were 14.2 ± 8.9 and 9.7 ± 6.3 in the first and second sessions, respectively. These results indicate that populations of follicles were increased till Day 11 after OPU, and proportion of normal oocytes were increased in the renewal follicles.
297 A CASE REPORT: THE OUTCOME OF IVP MAY BE RELATED TO THE BATCH OF TCM-199 IN IVM
Authors: Räty M. , Ketoja E. , Kananen-Anttila K. , Korhonen K. , Matomäki J. , Halmekytö M. , Peippo J.
Published: December 2004

Chemicals used in bovine IVP may be inhibitory to embryo development (see, e.g., Van Soom A et al. 1994 Theriogenology 41, 325). The present study continued earlier experiments to optimize serum-free IVM protocol for bovine oocytes (Räty M. 2004 Reprod. Fert. Dev. 16, 281). The embryo development rate was much lower in the new experiments than in our previous studies. Thorough testing indicated that the batch of TCM-199 medium used in maturation was the reason for the lower embryo development results. In total, 14,589 abattoir-derived bovine oocytes in 23 batches were matured for 24 h in TCM-199 with glutamax-I (GIBCO, Paisley, UK), 0.25 mM Na-pyruvate, 100 IU mL−1 penicillin, 100 μg mL−1 streptomycin, 10 μg mL−1 LH, 2 μg mL−1 FSH, and 1 μg mL−1 β-estradiol supplemented as follows: (1) 10% FBS (GIBCO, New Zealand); (2) 4 mg mL−1 fatty acid-free albumin (FAFBSA) (Sigma-Aldrich, Helsinki, Finland); (3) 4 mg mL−1 polyvinylpyrrolidone (PVP) (Sigma) + growth factors (GF; 100 ng mL−1 IGF-I + 100 ng mL−1 EGF); or (4) as (3) but without FSH, LH, and β-estradiol (PVPGFwoH). Fertilized oocytes were denuded and cultured in modified SOFaaci + 4 mg mL−1 FAFBSA in 5% O2. (Holm P. et al. 1999 Theriogenology 52, 683–700). Two TCM-199 batches were used in separate IVM runs, batch 1 (Lot#3075638; 10 runs) and batch 2 (Lot#3081334; 13 runs). The statistical analyses were based on generalized linear mixed models. The estimated probabilities for embryo cleavage and development are shown in Table 1. The use of TCM-199 batch 1 resulted in significantly lower embryo development rates than the use of TCM-199 batch 2 in every IVM group studied. PVP IVM groups were the most sensitive for the TCM-199 batch and resulted in reduced embryo cleavage as well as strikingly low Day 7 embryo development. Our results indicate that TCM-199 batch 1 disturbed embryo development. In defined IVM groups (PVP groups), the maturation inhibiting role of TCM-199 batch 1 was seen already at the cleavage stage. It is possible that FBS and FAFBSA may have protected the oocytes to some extent against inhibitory effects of TCM-199. There is evidence for BSA acting as a chelating agent (see, e.g., Flood L.P. and Shirley B. 1991 Mol. Reprod. Dev. 30, 226–231).

Table 1.
Estimated probability for embryo cleavage at 38–42 hpi and Day 7 embryo development after IVM in two different batches of TCM-199
299 IN VITRO DEVELOPMENT OF IMMATURE PORCINE OOCYTES FERTILIZED IN VITRO TO THE BLASTOCYST STAGE
Authors: Somfai T. , Kikuchi K. , Medvedev S.Y. , Onishi A. , Iwamoto M. , Fuchimoto D.-I. , Ozawa M. , Noguchi J. , Kaneko H. , Bali Papp A. , Sato E. , Nagai T.
Published: December 2004

In vitro fertilization (IVF) and embryonic development of mature and meiotically arrested porcine oocytes were compared in this study. After in vitro maturation (IVM) for 48 h of cumulus-oocyte complexes, 75.4% (n = 442) of them extruded a visible polar body (PB). Most oocytes with a polar body (PB+ group) were found to be at metaphase II (M-II) stage (91.4%). Most oocytes without a visible polar body (PB− group, n = 144) appeared to be arrested at the germinal vesicle (GV) (41.6%) and first meiotic metaphase (M-I) (34.0%) stages. After IVF of oocytes (the day of IVF = Day 0), there was no significant difference between PB+ and PB− groups in rates of sperm penetration, monospermy, and oocyte activation after the penetration. Embryonic development was assessed by staining with 1% orcein. On Day 2, although there was no difference between the embryo cleavage in PB+ (n = 447) and PB− (n = 217) groups (47.0% and 35.9%, respectively), PB+ embryos had more cells than the PB− embryos (3.37 and 2.81 cells, respectively) (P < 0.05; ANOVA). On Day 4, the cleavage rate of PB+ embryos was higher than that of PB− embryos (45.4% and 24.3%, respectively), and PB+ embryos had more cells than the PB− embryos (8.26 and 6.0 cells, respectively) (P < 0.05; ANOVA). On Day 6, a significantly higher number of PB+ embryos developed to the blastocyst stage than that of the PB− embryos (34.6% and 20.7%, respectively) (P < 0.05). However, by subtracting the GV oocytes from the PB− group, there was no difference in blastocyst rates between the M-I arrested and M-II oocytes (35.3% and 34.6%, respectively). The number of blastomer nuclei in embryos obtained from the PB+ group (52.0) was significantly higher than that of the PB− group (29.1); however, the proportion of inner cell mass and trophectoderm cells in PB+ and PB− blastocysts did not differ significantly (1:1.9 and 1:2.2, respectively) (P < 0.05). Chromosome analysis revealed that PB+ blastocysts had significantly more diploid blastomeres (69.7%) than PB− blastocysts (44.0%), whereas PB− blastocysts had significantly more triploid cells (34.0%) compared with PB+ oocytes (8.4%)(P < 0.05; χ2 test). These results indicate that porcine oocytes arrested at the M-I stage undergo cytoplasmic maturation during culture and have the same ability to develop to blastocysts after IVF as M-II oocytes but with a lower cell number; the latter might be caused by the slower embryonic development.
336 COMPARISON OF FETAL DEVELOPMENT AND FETAL HEARTBEAT BY ULTRASONOGRAPHY IN ARTIFICIALLY INSEMINATED AND EMBRYO-TRANSFERRED DAIRY COWS
Authors: Kishida K. , Shibata N. , Hata K. , Aoki S. , Nishisouzu T. , Dochi O. , Koyama H.
Published: December 2004

Bovine fetal size and fetal heartbeat are important factors for detecting abnormal pregnancies in artificially inseminated and embryo-transferred dairy cows. The purpose of this study was to compare fetal growth and fetal heartbeat by transrectal ultrasonography in the early pregnancy of artificially inseminated (AI) and embryo-transferred (ET) dairy cows. The pregnancies of 86 animals were examined by fetal heartbeat count (n = 35 per animal of AI versus n = 51 per animal of ET) every 5 days from 35 to 60 days after breeding (the day of ovulation = day 0). The pregnancies of 237 animals were measured by fetal crown-rump length (n = 95 per animal of AI versus n = 142 per animal of ET) at 30 days and 60 days after breeding. Built-in electronic calipers were used to measure fetal dimensions on the ultrasonic screen. In addition, the times of first fetal heartbeats were recorded. Data were analyzed using ANOVA. The results are presented in the Table (Values were mean ± SD except for No. of animals). There were no differences in the range of fetal heartbeat for AI and ET (161.0 to 185.3 beats min−1 versus 169.7 to 184.6 beats min−1). However, fetal heartbeats of AI at 35 days of gestation was higher (P < 0.05) than those in cows at days 40, 45, 50, 55 and 60 of gestation (185.3 ± 8.7 beats min−1 versus 161.0 ± 9.5, 165.0 ± 19.5, 170.3 ± 26.8, 169.4 ± 27.8, and 171.2 ± 11.9 beats min−1, respectively). There were no differences in fetal crown-rump length between AI and ET. The prediction equation for crown-rump length in relation to fetal age of AI was y = 0.0046x2 − 0.2452x + 4.4424, R2 = 0.9643, while that for ET was y = 0.0049x2 − 0.2699x + 4.9396, R2 = 0.9728. Crown-rump length at 30 days and 60 days of gestation did not differ between AI and ET (1.2 ± 0.1 cm versus 1.2 ± 0.1 cm, 6.4 ± 0.3 cm versus 6.5 ± 0.4 cm, respectively). In conclusion, there were no differences in fetal development and heartbeats between AI and ET dairy cows. In addition, there were no differences in fetal crown-rump length between AI and ET dairy cows.

Table 1.
Comparison of fetal development and heartbeat by ultrasonography in AI and ET dairy cows
63 IMPROVED IN VITRO DEVELOPMENT OF PORCINE EMBRYOS PRODUCED BY NUCLEAR TRANSFER, IVF AND PARTHENOGENESIS
Authors: Sage D. , Hassel P. , Petersen B. , Mysegades W. , Westermann P. , Lucas-Hahn A. , Niemann H.
Published: December 2004

Porcine nuclear transfer (NT) is an inefficient process and it is necessary to use as many as 120 NT embryos for each foster mother to obtain small litters of live piglets. In these experiments, we evaluated the effects of culture atmosphere and medium on the development of NT embryos by monitoring blastocyst rate and cell number of Day 6 blastocysts. Age matched IVF and parthenogenetic embryos were also evaluated for comparison. For all experiments a pool of oocytes was aspirated from ovaries collected in a local abattoir. Following aspiration, oocytes were allowed to mature for 40 h in North Carolina State University (NCSU)-37 medium (supplemented with cAMP and hCG/eCG for the first 22 h). After removal of the cumulus cells, denuded oocytes with polar bodies were selected for NT, enucleated, fused with fetal fibroblasts, and sequentially activated electrically and chemically by 3 h of treatment with 6-dimethylaminopurine (6-DMAP). A second group of oocytes from the same denuded pool were maintained in TL-HEPES medium and activated in parallel with the NT group to produce parthenogenetic embryos. A third group was fertilized with frozen-thawed epididymal semen and co-cultured for ∼12 h to give IVF embryos. All three treatment groups were subdivided into a control subgroup and an experimental subgroup. In the first experiment, we compared the effects of atmosphere (20% vs. 5% oxygen) on in vitro embryonic development in NCSU-23 medium. In the second experiment, we used only the 5% oxygen concentration and compared different culture media. One subgroup was maintained in standard NCSU-23 medium and the second subgroup was cultured in a two-step system for the first 58 h in modified NCSU-23 (without glucose but supplemented with 2.0 mM lactate and 0.2 mM pyruvate), followed by addition of glucose to give a final concentration of 5.55 mM. Data were statistically analyzed by analysis of variance and chi square test. Blastocyst rate and mean cell number in all three embryo groups were improved under 5% oxygen. The most dramatic effect was observed in the NT group, in which the blastocyst rate increased significantly (P < 0.001) from 6.7% ± 5.9 (n = 279) to 19.6% ± 8.9 (n = 250) and mean cell number increased from 17.7 ± 12.1 to 25.8 ± 10.3 cells per blastocyst. With 5% oxygen there was also an increase of blastocyst rates and mean cell numbers in both IVF and parthenogenetic groups. In the second experiment, blastocyst rate for NT embryos increased significantly (P < 0.05) from 21.8% ± 7.6 (n = 242) in conventional NCSU-23 to 31.5% ± 11.0 (n = 271) in the modified system whereas there was almost no difference in the mean cell number of both groups (29.2 ± 4.3 vs. 31.5 ± 5.3). In the groups of IVF and parthenogenetic embryos no difference was found. These results indicate that both the reduced oxygen and the modified culture medium are important for pre-implantation development of porcine nuclear transfer embryos.
80 GENE EXPRESSION PROFILES AND IN VITRO DEVELOPMENT FOLLOWING VITRIFICATION OF PRONUCLEAR AND 8 CELL-STAGE MOUSE EMBRYOS
Authors: Boonkusol D. , Baji Gal A. , Bodo S.Z. , Gorhony B. , Pavasuthipaisit K. , Dinnyes A.
Published: December 2004

The analysis of differences in gene expression of embryos in response to cryopreservation may explain some of the observed differences in further development. Experiments were conducted to study effects of two vitrification methods, solid surface (SSV) and in-straw vitrification (STR), for pronuclear and 8 cell-stage mouse embryos with regard to gene expression and in vitro development. Both stages of embryos were vitrified by SSV (Dinnyes et al. 2000 Biol. Reprod. 63, 513–518) using 35% ethylene glycol (EG) for vitrification solution (VS) and STR using 40% EG for VS. Warming and rehydration of embryos in the SSV method was performed by placing vitrified droplets into a 37°C 0.3 M trehalose solution for 1 min, and then for 2 min in each of 0.15 M trehalose, 0.075 M trehalose, and base medium at room temperature before culture in CZB medium. Warming of embryos for the in-straw method was performed by holding straws in air for 10 s and then plunging them into 37°C water for 15–20 s. The contents of the straws were expelled into 37°C 0.3 M trehalose solution and embryos were treated as above. No significant differences were found between immediate survival rates of embryos vitrified by SSV and STR in both stages. Blastocyst rates were significantly higher with SSV than with STR and not significantly different from those of the control (Table 1). These results show that SSV was more efficient than STR. Quantification of selected gene transcripts from single embryo (6 embryos/treatment group) was carried out by quantitative real-time RT-PCR. Genes related to oxidative stress (MnSOD and CuSOD), cold stress (CIRP, RBM3, and p53), and β-actin as reference gene were amplified. We found up-regulation of all stress genes at 3 h post-warming in all treatment groups. At 10 h post-warming, a low level of gene expression was found in SSV-treated embryos, but gene expression remained at high level in STR-treated embryos. However, no differences in gene expression were found between blastocysts developed from fresh and vitrified embryos. In conclusion, the real-time RT-PCR method from single embryos opened new opportunities for understanding molecular events following cryopreservation. The continuous up-regulation of stress-related genes at 10 h post-warming might have been an early indicator of reduced viability following STR, as was also indicated by the developmental rate to the blastocyst stage.

Table 1.
Survival and blastocyst rates of vitrified/warmed pronuclear and 8 cell-stage mouse embryos

This work was supported by the Thailand Research Fund (Royal Golden Jubilee Ph.D. scholarship) and a Bio-00017/2002 grant of the Hungarian National Office of Research and Technology.
97 ASSESSMENT OF VIABILITY OF IN VITRO PRODUCED BOVINE EMBRYOS FOLLOWING VITRIFICATION BY CVM OR SLOW FREEZING WITH ETHYLENE GLYCOL AND TRIPLE TRANSFER
Authors: Peachey B. , Hartwich K. , Cockrem K. , Marsh A. , Pugh A. , van Wagtendonk J. , Lindemans W.
Published: December 2004

Vitrification has become the method of choice for the preservation of in vitro derived embryos of a number of species, and several methods of vitrification have been developed. One such method, the cryoLogic vitrification method (CVM) yields high survival rates of warmed embryos (Lindemans W et al. 2004 Reprod. Fertil. Dev. 16, 174 abst). In this study, the post-warm viability of bovine IVP embryos following either vitrification using CVM or slow freezing using ethylene glycol (EG) was compared. In addition, the survival of embryos following triple transfer to synchronized recipients was measured and the embryo (“e”) and recipient (“r”) contributions to embryo survival was determined using the “er” model for embryo survival (McMillan WH et al. 1998 Theriogenology 50, 1053–1070). Bovine IVP methods were those of van Wagtendonk et al. 2004 Reprod. Fertil. Dev. 16, 214 (abst). On day 7 of culture (Day 0 = IVF), Grade 1 and 2 embryos that had reached at least the late morula stage were selected for vitrification (20% DMSO, 20% ethylene glycol) or freezing in 1.5 M ethylene glycol + 0.1 M sucrose (0.5°C/min to −35°C). Following storage in LN2 for at least 24 h the embryos were thawed, the cryoprotectant removed, and the embryos cultured for 72 h in mSOF medium under 5% CO2, 7% O2, 88% N2. The number of hatching embryos was recorded at 24-h intervals. In addition, blastocyst and expanded blastocyst embryos were thawed and immediately transferred nonsurgically to recipients (three embryos of the same grade to each recipient) on Day 7 of a synchronized cycle (Day 0 = heat). The recipients were ultrasound-scanned for the presence of, and number of, fetuses on Days 35 and 62, respectively. The invitro assessment of 148 CVM and 230 EG frozen embryos indicated that more vitrified than EG embryos hatched by 72 h (73% vs. 62%; CVM vs. EG, χ2 = 4.5, P < 0.05). Overall, more Grade 1 embryos hatched than Grade 2 (74% vs. 60%, χ2 = 7.2, P < 0.01). CVM embryos (105) were triple-transferred to 35 recipients, and EG embryos (30) were triple-transferred to 10 recipients. Recipient pregnancy rates at Day 62 were 80% and 70%, respectively. Overall embryo survival was 38.5% (41% for CVM and 30% for EG). The overall calculated “e” and “r” values were 0.39 and 1.0 (“e”: 0.42 and 1.0, and “r”: 0.31 and 1.0, respectively, CVM and EG groups). Survival rates of CVM embryos to Day 62 (41%) were slightly lower than that previously obtained for fresh embryos produced using an identical IVP procedure (44% – van Wagtendonk AM 2004).