bluuebunny/crossref_metadata_2025 huggingface dataset:
Freehand Biopsy Guided by Electromagnetic Needle Tracking: A Phantom Study
Authors: Ewertsen C. , Rue Nielsen K. , Bachmann Nielsen M.
Published: December 2011

Ziel: Bewertung der Genauigkeit und des Zeitaufwands bei einer Biopsie mittels elektromagnetischen Nadeltrackings an einem Phantom im Vergleich zur Standardtechnik der ultraschallgestützten Biopsie mit angeschlossener Lenkeinrichtung. Darüber hinaus die Bewertung der Biopsie außerhalb der Ebene gestützt duch Nadeltracking.

Material und Methoden: An einem Phantom wurden drei verschiedene Biopsieserien durchgeführt: eine, bei der die Lenkeinrichtung an den Meßwandler ohne Nadeltracking angeschlossen ist und somit der Standardtechnik der ultraschallgestützten Biopsie ähnelt (Serie 1), eine Freihand innerhalb der Scan-Ebene mittels elektromagnetischen Nadeltrackings (Serie 2) und eine Freihand außerhalb der Scan-Ebene mittels elektromagnetischen Nadeltrackings (Serie 3). Das Phantom enthielt Kugeln von 1 cm Durchmesser, die mit roter Farbe gefüllt waren. Jede Perforation der Phantomoberfläche wurde als eine Biopsie gewertet.

Ergebnisse: 180 Biopsien wurden durchgeführt. Die mittlere Zeitdauer pro Biopsie betrug in Serie eins 19,9 s (SD: 9,1), in Serie zwei 34,1 s (SD 17,9) und in Serie drei 34,4 s (SD: 14,0). Die Erfolgsrate betrug insgesamt: 88 % für die nadelgeführte Biopsie in der Ebene, 87 % für das Nadeltracking in der Ebene und 92 % für das Nadeltracking außerhalb der Ebene. Zwischen den Methoden konnte kein statistisch signifikanter Unterschied ausgemacht werden.

Schlussfolgerung: Die Nadelnavigation ist ein möglicherweise wertvolles Hilfsmittel der bildgestützten Biopsie mit einer ähnlichen Erfolgsquote im Vergleich zur konventionellen bildgestützten Biopsie. Darüber hinaus ermöglicht sie die bildgestützte Biopsie außerhalb der Ebene.
Structural and thermoelectric properties of BaRCo4O7 (R = Dy, Ho, Er, Tm, Yb, and Lu)
Authors: Wong-Ng W. , Xie W. , Yan Y. , Liu G. , Kaduk J. , Thomas E. , Tritt T.
Published: December 2011

The structure and thermoelectric properties of a series of barium lanthanide cobaltites, BaRCo4O7 (R = Dy, Ho, Er, Tm, Yb, and Lu), which were prepared using the spark plasma synthesis technique, have been investigated. The space group of these compounds was re-determined and confirmed to be P31c instead of the reported P63mc. The lattice parameters a and c range from 6.26279(2) Å to 6.31181(6) Å, and from 10.22468(6) Å to 10.24446(15) Å for R = Lu to Dy, respectively. The crystal structure of BaRCo4O7 is built up from Kagome sheets of CoO4 tetrahedra, linked by triangular layers of CoO4 tetrahedra. The values of figure of merit (ZT) of the BaRCo4O7 samples were determined to be around 0.02 at 800 K. X-ray diffraction patterns of these samples have been determined and submitted to the Powder Diffraction File.
Electrically tuneable spectral responsivity in gated silicon photodiodes
Authors: Abid Kamran , Wang Xingsheng , Khokhar Ali Z. , Watson Scott , Al-Hasani Sulaiman , Rahman Faiz
Published: December 2011

We report the observation of electrically tuneable spectral responsivity in silicon-based photodetectors. The current flowing through a lateral p-i-n junction photodiode can be changed by changing either the gate bias or the intensity of incident light, with the devices exhibiting typical optical responsivities of 65 A/W. The peak sensitivity of the device can be changed over the entire visible region by changing the gate voltage in a 5 V range. This happens because with increasing gate bias, an accumulation layer of holes is pulled closer to the Si-SiO2 interface, enhancing the blue response of the device.
Study of surface passivation of crystalline silicon with amorphous silicon carbide deposited by plasma enhanced chemical vapor deposition
Authors: Klein D. , Kunst M.
Published: December 2011

Electrical passivation induced by a silicon carbide coating deposited on mono-crystalline silicon substrate was investigated by means of photo-conductivity measurements. Roles of the fixed charges and surface defects were compared with silicon nitride coating. The passivation mechanisms were found to be the same as for silicon nitride but a smaller number of fixed charges in the range of the silicon oxide was calculated.
Surface effects in a semiconductor photonic nanowire and spectral stability of an embedded single quantum dot
Authors: Yeo Inah , Malik Nitin S. , Munsch Mathieu , Dupuy Emmanuel , Bleuse Joël , Niquet Yann-Michel , Gérard Jean-Michel , Claudon Julien , Wagner Édouard , Seidelin Signe , Auffèves Alexia , Poizat Jean-Philippe , Nogues Gilles
Published: December 2011

We evidence the influence of surface effects for InAs quantum dots embedded into GaAs photonic nanowires used as efficient single photon sources. We observe a continuous temporal drift of the emission energy that is an obstacle to resonant quantum optics experiments at the single photon level. We attribute the drift to the sticking of oxygen molecules onto the wire, which modifies the surface charge and hence the electric field seen by the quantum dot. The influence of temperature and excitation laser power on this phenomenon is studied. Most importantly, we demonstrate a proper treatment of the nanowire surface to suppress the drift.
Control of random lasing in ZnO/Al2O3 nanopowders
Authors: Nakamura Toshihiro , Tiwari Bishnu P. , Adachi Sadao
Published: December 2011

We demonstrate that the random lasing wavelength of ZnO/Al2O3 nanopowders can be controlled by varying the weight fraction of Al2O3. This controllability is due to the fact that the extent of self-absorption by ZnO nanopowder can be modified by changing the photon-transport mean free path. The lasing threshold excitation power is also dependent on the weight fraction of Al2O3. The random lasing characteristic can be explained well by a theoretical model based on the photon-transport mean free path.
Two-photon focal modulation microscopy in turbid media
Authors: Si Ke , Gong Wei , Chen Nanguang , Sheppard Colin J. R.
Published: December 2011

The image formation of two-photon focal modulation microscopy (2PFMM) in turbid media is theoretically investigated. The results show that compared with conventional two-photon fluorescence microscopy, the ballistic excitation of 2PFMM is concentrated in a much smaller region around the focal point and decays more rapidly outside the focal volume, while the scattered excitation is largely suppressed. When focuses at 1600 μm, the signal-to-background ratio and signal-to-noise ratio of 2PFMM are improved by 30 dB and 18 dB, respectively, indicating that 2PFMM can achieve a large imaging penetration depth.
Electric field controlled magnetization rotation in exchange biased antiferromagnetic/ferromagnetic/piezoelectric composites
Authors: Lebedev G. A. , Viala B. , Lafont T. , Zakharov D. I. , Cugat O. , Delamare J.
Published: December 2011

Electric field controlled rotation of the magnetization direction of a ferromagnet is investigated in the composite of antiferromagnetic (AF)/ferromagnetic (F)/piezoelectric (PE) layers. Usually, simple 90° magnetization switching is reported with uniaxial F/PE composites. In contrast, when considering unidirectional anisotropy in exchange-biased AF/F systems, a controllable rotation of the magnetization direction in F can be achieved. Here, we show a composite which differs by using in-plane d33 deformation mode with standard magnetic electrodes. We report the experimental evidence of magnetization continuous rotation in IrMn/CoFeB/PZT composites leading to technological impacts on memories, sensors, and microwave devices.
Ballistic thermal conductance in graphene nanoribbon with double-cavity structure
Authors: Peng Xiao-Fang , Wang Xin-Jun , Gong Zhi-Qiang , Chen Ke-Qiu
Published: December 2011

We investigate phonon transport and thermal conductance in a Graphene Nanoribbon modulated with a double-cavity quantum structure at low temperatures. Two methods are compared: the force-constant and elastic wave continuum models. Calculations show that both the models show the similar thermal conductance property at low temperatures despite the excited theory of the discrete phonon modes in quantum structure being not the same. However, in the higher temperature region, the thermal conductance in the force-constant model is bigger than that in the elastic wave continuum model. The difference originates from the inequable cutoff frequencies of the phonon modes. A brief analysis of these results is given.
The physiology of multifactorial problems limiting the establishment of pregnancy in dairy cattle
Authors: Evans Alexander C. O. , Walsh Siobhan W.
Published: December 2011

The failure of cows to successfully establish pregnancy after insemination is an important limiting factor for the efficiency of dairy production systems. The physiological reasons for this are many and pertain to the post partum and early pregnancy periods. Cows that suffer severe negative energy balance after parturition are prone to diseases (including uterine infection) that are, in part, explained by reduced function of the immune system, having negative consequences for subsequent fertility. In high-producing dairy cows, the duration and intensity of oestrus is low as a consequence of low circulating oestradiol concentrations, and after insemination, high embryo mortality is the single biggest factor reducing calving rates. Embryo mortality occurs as consequences of poor oocyte quality (probably caused by the adverse metabolic environment) and by poor maternal uterine environment (probably caused by carry-over effects of uterine infection and low circulating progesterone concentrations). Immediate improvements in the fertility of lactating cows on many farms can be achieved by applying existing knowledge, but longer-term sustained improvement will require additional knowledge in many areas including the physiology of the tissues that contribute to reproduction.
The effects of endometritis on the establishment of pregnancy in cattle
Authors: Gilbert Robert O.
Published: December 2011

Endometritis is common in post partum dairy cows and is associated with impaired reproductive performance reflected in reduced first service conception, reduced hazard of pregnancy over the breeding period and increased risk of reproductive culling. The observed effects may be mediated directly by bacterial products, such as lipopolysaccharide (LPS, endotoxin), or indirectly by inflammatory mediators, such as cytokines, eicosanoids, nitric oxide and oxidative stress affecting sperm, ovarian, uterine and embryonic function. An inflammatory milieu in the uterus has been associated with changes in sperm motility and function as well as increased sperm phagocytosis. Zygotes resulting from fertilisation of oocytes with sperm subjected to oxidative stress are less likely to develop to the blastocyst stage. In addition, LPS and tumour necrosis factor-α (TNFα) impair follicular steroidogenesis, growth and ovulation. Oocytes exposed to LPS or prostaglandin (PG) F2α during maturation are less likely to develop to blastocyst stage after fertilisation. Embryos exposed to inflammatory mediators during development have fewer trophoectoderm cells. Nitric oxide impairs development of preimplantation embryos and TNFα increases blastomere apoptosis. Endometritis in women has been associated with higher rates of implantation failure. Extragenital inflammation (e.g. mastitis) is also associated with an increased rate of embryonic loss in cattle. These observations make it clear that direct and indirect effects of endometritis, and inflammation in general, can interrupt successful reproduction at several crucial stages.
102 ACCURACY OF PRE-IMPLANTATION GENETIC DIAGNOSIS USING CELLS BIOPSIED FROM EQUINE BLASTOCYSTS
Authors: Choi Y. H. , Penedo M. C. T. , Daftari P. , Velez I. C. , Hinrichs K.
Published: December 2011

There is growing interest in pre-implantation genetic diagnosis (PGD) for management of inherited genetic disease in the horse. In a previous study (Choi et al. 2010 Reproduction 140, 893–902), we demonstrated normal viability of equine blastocysts after biopsy. However, genome amplification was only moderately successful and only 1 of 2 analyses of heterozygous loci accurately detected both alleles. In the current study, we investigated different methods for amplification of DNA to improve the efficiency of PGD. To evaluate allele drop-out, multiple commonly-heterozygous gene loci were evaluated. In Experiment 1, using a piezo drill, 3 to 5 biopsy samples of 20 to 30 cells each were obtained from each of 4 in vitro-produced blastocysts. The samples and embryos were stored at –20°C, then shipped to the Veterinary Genetics Laboratory at the University of California, Davis. Whole genome amplification was done with an Illustra Genomiphi V2 kit (GE Healthcare, Waukesha, WI) before PCR for specific markers. Two disease-related (SCN4A and PPIB), one gender (AME) and 17 microsatellite identification markers were genotyped, for a total of 20 loci. Results for biopsy samples were compared with those for the corresponding embryo. A DNA signal was obtained from 14/15 biopsy samples, but for only 59.6% of the 280 total genotypes. Of 40 heterozygous loci, the signal from the corresponding biopsy sample showed only one allele (underwent allele dropout) in 60/80 instances (75%). In Experiment 2, 4 biopsies were obtained from each of 4 additional in vitro-produced blastocysts, then all samples were stored at –20°C. The Repli-G Mid kit (Qiagen, Valencia, CA) was used for whole genome amplification. Two disease-related (SCN4A and PPIB), 2 gender (AME and eSRY), 10 coat colour and 17 identification markers (total of 31 loci) were examined in each biopsy sample and were compared with results for the embryos. One biopsy sample was lost. Signal was obtained from 14/15 of the remaining biopsy samples and gave a 100% match at the 2 gender loci, 2 disease-related loci and 10 coat colour loci. One identification locus, LEX33, amplified in only 8 of 22 analyses. At the remaining 16 identification loci, 223/224 biopsy results matched those for the embryos. Overall, of 51 heterozygous loci among the 4 embryos, biopsy samples exhibited allele dropout in 1/180 instances (0.6%). In conclusion, results obtained using piezo-driven embryo biopsy and whole genome amplification using the Qiagen Repli-G kit have high accuracy and this technique may be suitable for use in a clinical setting. Further studies are needed with in vivo-derived embryos and to optimize accuracy of PCR of some identification markers.
This work was supported by the American Quarter Horse Foundation, the Link Equine Research Endowment Fund, Texas A&M University and by Ms Kit Knotts.
104 BIRTH OF FIRST BUFFALO (BUBALUS BUBALS) CALF FOLLOWING EMBRYO SPLITTING AND POLYMERASE CHAIN REACTION SEXING AT BLASTOCYST STAGE
Authors: Zhang M. , Chen H. H. , Tang J. W. , Liang X. W. , Chen M. T. , Qin G. S. , Fu Q. , Zhang X. F. , Zheng H. Y. , Lu K. H.
Published: December 2011

Embryo-splitting technology provides an effective procedure for increasing the number of transferable embryos per donor, producing genetically identical offspring and facilitating embryo sexing. The ability to identify the sex of embryos before transfer will offer a reliable, economical and practical procedure for buffalo breeding. In this study, we have assessed the feasibility of production of offspring with controlled sex in buffalo by first comparing the effect of blastocyst quality on the viability of demi-embryos and then identifying the sex of a demi-embryo by multiplex-nested PCR before transfer into the recipient. In vitro-matured buffalo oocytes were fertilized by IVF and cultured to the blastocyst stage for 6 to 7 days as described by Lu et al. (2007 Anim. Reprod. Sci. 100, 192–196). These blastocysts were classified in terms of their developmental pattern and morphology on a scale of 1 to 3 grades as described by McEvoy et al. (1990 Theriogenology 33, 1245–1253). Blastocysts were split into 2 equal parts by a micromanipulation system. Viability of the resulting demi-embryos was confirmed by formation of a blastocoel cavity and definite inner cell mass after culture for 24 h. One of the zone-free demi-embryos derived from a grade-1 blastocyst was cultured in TCM 199 supplemented with 10% fetal bovine serum for another 2 h, then was transplanted to a spontaneous oestrous recipient. The other demi-embryo was used for sexing by multiplex-nested PCR (Fu et al. 2007 Theriogenology 68, 1211–1218). The results showed that grade-1 blastocysts yielded more viable demi-embryos than grade-2 and grade-3 blastocysts [P < 0.01; 73/92 (79.67%) vs 32/76 (47.05%) vs 26/94 (26.53%), respectively]. Transplantation of the presumed-Y demi-embryo derived from grade-1 blastocyst into a recipient resulted in the birth of a male buffalo calf. To the best of our knowledge, this is the first buffalo calf produced following embryo splitting and PCR sexing of the embryo at the blastocyst stage. Successful birth of the desired-sex offspring in the present study indicates the feasibility of using embryo splitting in combination with multiplex-nested PCR sexing to produce offspring of controlled sex in swamp buffalo. However, the quality of embryos before splitting was an important factor governing the in vitro development of viable demi-embryos.
This study was supported by the Guangxi Science and Technology R&D Program (0626001-3-1, 0815008-2-4).
105 BOVINE EMBRYO TYPE, GRADE AND RECIPIENT SYNCHRONY, BUT NOT CORPUS LUTEUM QUALITY, INFLUENCED PREGNANCY RATES IN COMMERCIAL EMBRYO TRANSFER
Authors: Burns G. , Long C. , Gibbons J. , Shull J.
Published: December 2011

Embryo transfer is a tool that enables progressive cattle producers to reach their reproductive, genetic and financial goals. The complexity of pregnancy establishment increases with any reproductive technology, especially with one with many variables. This clinical analysis employed a statistical model capable of explaining factors that affect pregnancy probability resulting from over 8000 embryo transfers. These data included corpus luteum (CL) quality score, embryo stage (morula to blastocyst) and grade (1–3), recipient synchrony (relative to the embryo age) and pregnancy rate (≥45 days). The CL score was an amalgamation of the palpable diameter of the CL, whether or not a crown was present and the overall quality of the reproductive tract (size of the uterine horns and perceived patency of the cervix). Embryos produced by IVF were transferred fresh and in vivo–produced embryos were transferred fresh (VIVO FRESH) or following cryopreservation (VIVO FROZ). Statistical methods were used to compare types of embryos transferred and pregnancy rates. All data were analysed using JMP 2009 software by logistic regression with the Logit link. Analysis for the odds of pregnancy occurring among groups using contrasts was conducted. Fisher's exact test showed significant differences (P < 0.05) among groups for pregnancy rate, with the VIVO FRESH embryos group having the highest rate (68.5 ± 1.5%) and the VIVO FROZ the lowest (54.7 ± 1.6%) and the IVF embryos were intermediate (62.1 ± 3.3%). Synchrony, stage, embryo grade and CL quality were analysed for their predictive ability of pregnancy rates in the IVF, VIVO FRESH and VIVO FROZ groups using effect tests with a P < 0.05. Interestingly, the CL quality had no predictive ability for any of the groups. Effectors that had an influence on the VIVO FROZ group included synchrony, stage and embryo grade. However, for the IVF and VIVO FRESH groups, pregnancy rates were affected by only embryo grade. Retrospective, observational analysis of commercial embryo transfer data represents a challenge to meaningful interpretation. Because of the nature of the business, experimental treatments cannot always be developed and implemented. Nonetheless, valuable information is gained by using appropriate statistical methods. As anticipated, these data suggested that the type of embryo transferred, grade and synchrony of the recipient are all important factors, particularly for cryopreserved embryos. Unexpectedly, the score of the CL did not influence pregnancy outcome regardless of embryo type. Evaluation of clinical data allows adjustment of embryo transfer protocols, facilitates genetic gain via enhanced reproductive success and provides a unique opportunity to analyse large, real-world data sets.
112 BLUETONGUE VIRUS INFECTION IN CATTLE AFTER TRANSFER OF BOVINE IN VIVO-DERIVED EMBRYOS
Authors: Vandaele L. , De Clercq K. , Van Campe W. , De Leeuw I. , Van Soom A.
Published: December 2011

Bluetongue virus (BTV) has been categorized by the OIE as a category 1 disease agent, for which proper handling between collection and transfer is thought to be sufficient to prevent transmission through embryo transfer. For bovine viral diarrhoea virus, it was shown that effectiveness of washing procedures depends on virus strains (Waldrop et al. 2004 Theriogenology 62, 45–55). Also BTV-8 has unique characteristics in comparison with other strains (De Clercq et al. 2008 Transbound. Emerg. Dis. 55, 352–359). The aim here was to investigate whether embryo transfer of in vivo-derived bovine embryos after in vitro exposure to BTV-8 can be performed without risk for infection of the recipients if IETS washing and trypsin treatment procedures are followed. Donor cows (n = 2) were synchronized and superovulated using Stimufol® (Ulg, Liége, Belgium) and subsequently inseminated. At 6.5 days post-insemination (dpi), flushed embryos (n = 14 and n = 3) were placed in 800 μL of minimal essential medium (MEM), containing 104.9 50% tissue culture infectious doses (TCID50) of BTV-8 (Bel 2006/2 P5, VAR, Brussels, Belgium) and incubated for 1 h at 39°C in 5% CO2 in air (Vandaele et al. 2011 Vet. Res. 42, 14–21). Next, embryos were washed in pairs in 5 consecutive Petri dishes containing PBS with antibiotics and 0.4% BSA, w/o Ca and Mg. Then, embryos were exposed to 2 consecutive trypsin (Invitrogen, Carlsbad, CA, 25050-014) washes of 45 s each at 39°C in 5% CO2 in air and finally, another 5 consecutive washes in PBS with 2% FCS. Each Petri dish contained at least 2 mL of medium and was gently agitated between washes. Embryos were transferred in a maximum of 7 μL of medium and a new tip was used after every wash step. Washes 1 to 5 and washes 6 to 10 were pooled and analysed for BTV-8 (RT-qPCR). After these washes, 3 pairs of embryos (n = 6) were loaded in straws and transferred to 3 BTV-8 negative recipients. Two sentinel cows served as control. Cows were bled twice weekly and blood and serum samples were analysed for BTV-8 (RT-qPCR) and BTV-8 antibodies. Viral BTV-RNA was detected in all 3 recipient cows at 7 days after transfer and viraemia was confirmed by the establishment of high antibody titers at 14 days after transfer. Viral BTV-RNA was detected in washes 1 to 5 for each pair of embryos (Cp-value around 29), whereas washes 6 to 10 had Cp-values around the cut-off value (40), indicating that probably the last wash was BTV-8 negative. None of the recipients was pregnant at 28 days post-transfer. In conclusion, washing and trypsin treatment did not succeed in removing BTV-8 from in vitro-spiked in vivo-derived bovine embryos. These unexpected results stress the need for further in vivo research, e.g. what is the virus load in vivo embryos may be exposed to in utero during viraemia? Does BTV-8 react differently with the zona compared with other strains? Are alternative washing procedures needed to remove BTV-8 from the zona?
114 LIVE-CELL IMAGING FOR THE QUALITY ASSESSMENT OF INTRACYTOPLASMIC SPERM INJECTION EMBRYO IN CYNOMOLGUS MONKEY
Authors: Morichika J. , Yamagata K. , Iwatani C. , Tsuchiya H. , Kusanagi A. , Wakayama T. , Torii R.
Published: December 2011

We have developed a live-cell imaging technique to assess measures of embryo quality such as epigenetic status and chromosome integrity during the early cleavage stages of pre-implantation development in the mouse. The advantages of this method are that the procedure is safe for the embryo and pups are not transgenic even after the imaging (Yamagata et al. 2009 Hum. Reprod. 24, 2490–2499). One of the valuable indexes in using this imaging technique is chromosome segregation (CS) during first mitosis; the embryos showing normal CS (NCS) result in normal offspring, whereas abnormal CS (ACS) embryos do not. In this study, we established a live-cell imaging technique for cynomolgus monkey intracytoplasmic sperm injection (ICSI) embryos and we succeeded in obtaining a normal offspring from NCS embryos after the assessment of live-cell imaging. Ovarian stimulation was carried out as previously described by Torii et al. (2001 Exp. Anim. 50, 259). Oocytes were collected by follicular aspiration using laparoscopy and ICSI was performed to metaphase II oocytes. After the ICSI, a mixture of mRNA encoding fluorescent labelled tubulin and histone was injected into ICSI embryos for the evaluation. Live-cell imaging was initiated 4 h after injection by laser confocal microscopy and 2-cell embryos were classified as NCS or ACS the next day. After embryo culture, embryo transfer (ET) was carried out to recipient donors (NCS embryos: 13, ACS embryos: 2) and pregnancy was diagnosed by ultrasonography at 4 weeks after ET. In another experiment, we tried to assess the 2-cell embryos with a snapshot image taken by a conventional fluorescent microscope as a simplified method. A total 121 embryos from 15 monkeys were analysed and embryos were classified as NCS or ACS. Live-cell imaging revealed that the NCS rate was 43.3% and the ACS rate was 56.7%. Pregnancy was confirmed in 2 NCS embryos from 13 ET (15.4%; 2/13); however, no pregnancy was observed in the ACS group (0%, 0/2). Furthermore, one normal offspring was achieved from ET of 2 NCS embryos that were diagnosed by live-cell imaging. In addition, we could also assess the status of chromosome and nuclei in the 2-cell embryos even by fluorescent microscopy and in this case, the NCS rate was 69.2% and the ACS rate was 30.8%. In conclusion, live-cell imaging can be used to evaluate the status of chromosome segregation of ICSI embryos in the cynomolgus monkey under laser confocal and fluorescent microcopy. The results indicate that ACS would be a detrimental factor in the embryonic development in the monkey, similar to in the mouse. Moreover, a normal offspring was born after the imaging and therefore this new technique could be applicable to assessment of embryo quality in human assisted reproductive technology.
135 BREEDING VALUES CONCORDANCE BETWEEN EMBRYOS AND CORRESPONDING CALVES
Authors: Le Bourhis D. , Mullaart E. , Schrooten C. , Fritz S. , Coppieters W. , Ponsart C.
Published: December 2011

Genomic tools are now available for most livestock species and are used routinely for genomic selection in cattle. Recently, biopsies of pre-implantation-stage embryos were genotyped for multiple markers. This strategy provides the opportunity to estimate breeding values for traits of particular interest and the presence of genetic abnormalities, thus allowing selection of embryos before transfer (Le Bourhis et al. 2011 Reprod. Fertil. Dev. 23, 197 abst). The present work aimed to compare the genotype and the breeding values from biopsied bovine embryos with the corresponding calves. Bovine embryos were obtained after superovulation (8 cows) and flushing at Day 6 or 7. A total of 11 embryos (1 or 2 Grade 1 embryos per flush per cow) were washed and biopsied using a microblade. Biopsies of 5 to 10 cells were transferred individually as dry samples in tubes and frozen before whole genome amplification (WGA). The genomic DNA of each biopsy was amplified using a WGA kit according to the manufacturer's instructions (WGA; QIAGEN REPLI-g® Mini Kit, Qiagen, Valencia, CA, USA). Biopsied embryos were transferred either frozen or fresh to synchronized recipients. At birth a blood sample was taken from the calf for subsequent genotyping. Genotyping was done using the Illumina BovineSNP50TM beadchip. Only embryos with call rate (CR) higher than 80% were selected for breeding values comparisons. Because of allele dropout, heterozygous markers are turned into homozygous makers artificially. Only markers that were still heterozygous in genotypes of an embryo were selected and error rates were calculated with the same markers from the corresponding calf. Imputation was done using Beagle and taking into account the 50 k genotyping results of the parents. Breeding values (milk production and morphological traits) were calculated and compared with those of the 7 corresponding calves. From 11 embryos analysed, only 1 (9%) gave a CR lower than 80% and a higher percentage error with calf genotype (Table 1). These results indicate that, using embryonic DNA after WGA, genotyping errors between embryo and calf are low and correlated with the CR of embryos. For embryos with CR higher than 84, the concordance of genomic values between embryo and calf is very high. More embryo–calf pairs are needed to assess the reliability of this method and to validate the breeding value at the embryo stage. This new technique offers new possibilities in managing breeding schemes by selecting the embryos before transfer.

Table 1.Embryo call rate (CR) and genotyping and breeding value concordances between embryos and calves
150 EFFECT OF MATERNAL OBESITY ON PERICONCEPTION FERTILITY AND BLASTOCYST GENE EXPRESSION IN THE MOUSE MODEL
Authors: Bermejo-Alvarez P. , Rosenfeld C. S. , Roberts R. M.
Published: December 2011

Obesity is frequently associated with infertility in humans and domestic animals. In this study, we aimed to study the effect of diet-induced obesity on oestrus cyclicity, ovulation rate, pre-implantation embryo development and blastocysts gene expression. Twenty-one NIH Swiss female mice were divided in 2 groups: one fed with a control diet (C, n = 9) and the other with a diet high in fat (F, n = 12) for 12 weeks. Dams were naturally bred and killed 3.5 days post-coitum. Bodyweight, numbers of corpora lutea and embryos and days required to breed the dams were recorded. Blastocysts were recovered and snap frozen in groups representing the individual dams. Expression of 10 candidate genes was analysed relative to H2afz. Data (means ± standard error of the means) were analysed by ANOVA (P < 0.05, significant). Five mice in the F group failed to breed during 20 days. These acyclic animals were significantly heavier than the other 7 of the F group (53.8 ± 3.8 vs 41.6 ± 0.9 g), with 4 weighting over 50 g. The seven cyclic animals in the F group showed a higher weight (41.6 ± 0.9 vs 31 ± 0.8 g) and higher number of days to mate (7.3 ± 1.6 vs 2.9 ± 0.5) compared with group C, but ovulation rates did not differ and reflected final blastocyst recovery. The expression level of 5 genes (Insr, Igf1r, Igf2r, Adipor1 and Adipor2) encoding for receptors of hormones related to metabolism regulation did not differ among groups. Two genes with roles in glucose (Scl2a1) and lipid transport (Ldlr) were down-regulated in the F group compared with the C group (Scl2a1: 1 ± 0.1 vs 1.38 ± 0.1; Ldlr: 1 ± 0.1 vs 1.24 ± 0.1); however, there were no differences in expression of the genes Gapdh (which encodes an essential glycolytic enzyme), Cpt1a (whose product catalyzes the rate limiting step of β-oxidation) and Sod2 (which encodes the mitochondrial isoform of superoxide dismutase). These results suggest that diet-induced obesity in the mouse model reduces fertility through oestrus cyclicity, blocking or prolonging the oestrus cycle, but there was no effect on ovulation rate or embryo development in those females that ovulated. The down-regulation of 2 genes related to nutrient uptake (Slc2a1 and Ldlr) in the F group suggests a protective mechanism to abate the excess of nutrients. Such protection may be effective, as the expression of genes for key metabolic markers for anaerobic glycolysis, fatty acid metabolism and oxidative stress remained unaltered.
Supported by Lalor Foundation to P. Bermejo-Alvarez, HD21896 to R. M. Roberts and RC1 ES018195 to C. S. Rosenfeld.
151 EVALUATION OF REFERENCE GENE TRANSCRIPT STABILITY DURING EARLY PRE-IMPLANTATION DEVELOPMENT AND IN SPERMATOGONIAL CELLS IN THE CAT
Authors: Biancardi M. N. , Pope C. E. , Powell R. H. , Galiguis J. , Dumas C. , Morris A. , Gómez M. C.
Published: December 2011

Differences in the stability of commonly used reference genes have been discovered between species and among different tissue types. These inconsistencies underscore the importance of validation and selection of an appropriate reference for normalization of gene expression to an endogenous control in RT-qPCR experiments. Three different reference transcripts (GAPDH, RPS19 and 18S rRNA) of 3 different functional categories were selected for evaluation during pre-implantation embryonic development and in spermatogonial cells in the domestic cat. Amplification efficiency was done by standard curve analysis with 5-fold template dilutions. For pre-implantation analysis, transcripts were assayed at 4 developmental stages: 2- to 6-cell (2–6C), 8- to 16-cell (8–16C), morula (M) and blastocyst (BL). Embryos were from pools of 4 to 10 in vitro matured and fertilized domestic cat embryos. For spermatogonia, isolates of 20 000 to 30 000 cells were assayed from single testis isolates following a collagenase and trypsin with DNase digestion and Percoll gradient separation. Total mRNA was isolated using the Cells-to-cDNA II Kit, with a minimum of 2 biological replicates for each sample type. The RNA quantitation was done by RiboGreen analysis and 47 ng of RNA was used for cDNA synthesis. Transcript abundance was detected in 2 technical replicates per sample by SYBR Green chemistry and data were analysed with NormFinder and one-way ANOVA. Each transcript showed varying levels of expression throughout development and among embryonic stages and in spermatogonial cells. The GADPH and 18S rRNA transcripts were expressed at higher levels in BL than in 2–6C and 8–16C, respectively. Contrarily, transcript levels of RSP19 were lower in BL compared with levels at 2–6C. Even though differences were observed among embryonic stages, analysis with NormFinder indicated that the most stable reference gene throughout early pre-implantation development was RPS19. When each stage was analysed separately, 18S rRNA was the most stable at 2–6C, whereas RPS19 was found to be the most stable at 8–16C, M and BL stages. In spermatogonial samples, GAPDH was the most stably expressed gene. Our results support the selection of an appropriate reference gene based on the needs of the experimental design. We conclude from these findings that when gene expression throughout the duration of early embryonic development is examined, RPS19 is the preferred selection. If gene expression is analysed within discrete time points of development only, it is appropriate to select 18S rRNA at the 2–6C stage and RPS19 for 8–16C, M and BL stage embryos. For spermatogonial samples, if comparing only different biological replicates, GAPDH should be selected for normalization of expression; however if the purpose of the experiment is to assay genes that are also expressed in BL, the most stably expressed reference gene is RPS19.
152 BISPHENOL A AND 17-BETA-OESTRADIOL RESULTED IN THE GENE ALTERATIONS IN OESTROGEN-RECEPTOR POSITIVE BG-1 OVARIAN CANCER CELLS
Authors: Hwang K.-A , Hyun S.-H. , Jeung E.-B. , Choi K.-C.
Published: December 2011

Because endocrine disrupting chemicals may interfere with the endocrine systems of our body and have an oestrogenic activity, we evaluated the effects of bisphenol A (BPA) on the transcriptional levels of altered genes in oestrogen receptor (ER)-positive BG-1 ovarian cancer cells. A microarray and RT-qPCR were employed to detect gene alterations in these cells following treatments. In this study, treatment with 17-β-oestradiol (E2) or BPA increased mRNA levels of E2-responsive genes related to apoptosis, cancer and cell cycle, signal transduction and nucleic acid binding and so on. Parallel with the microarray data, the mRNA levels of some altered genes including RAB31_member RAS oncogene family (U59877), cyclin D1 (X59798), cyclin-dependent kinase 4 (U37022), IGF-binding protein 4 (U20982) and anti-mullerian hormone (NM_000479) were significantly induced by E2 or BPA in this cell model. These results indicate that BPA in parallel with E2 induced the transcriptional levels of E2-responsive genes in an ER-positive BG-1 cells. In conclusion, these microarray and RT-qPCR results indicate that BPA, a potential weak oestrogen, may have an oestrogenic effect by regulating E2-responsive genes in ER-positive BG-1 cells and that BG-1 cells would be the best in vitro model to detect these oestrogenic endocrine disrupting chemicals.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011-0015385).
16 EFFECT OF DONOR CELL AND TRICHOSTATIN A ON DEVELOPMENT OF CLONED DAIRY CATTLE EMBRYOS
Authors: Mei-Ling Z. , Yun-Hai Z. , Yong T. , Ya L. , Hong-Guo C. , Yun-Sheng L. , Oto H. , Josef K. , Xiao-Rong Z.
Published: December 2011

The objective of present study was to investigate the effects of treatments to donor cells with fresh digestion (FD), cryopreservation/thawing (CT), trichostatin A (TSA) and durations of culture using TSA-CR1aa medium on in vitro development of dairy cow cloned embryos. In addition, some somatic cell cloned embryos were transferred to surrogates in heat to evaluate the in vivo developmental competence. The results (Table 1) showed that pretreatment of donor cells using TSA could significantly increase both cleavage and blastocyst rates of embryos (P < 0.05) compared with FD and CT group, whereas no significant difference was found between FD and CT group. When cloned embryos were subjected to TSA treatment in CR1aa for different times (0, 24, 48 and 60 h), the results showed that the blastocyst rate in the 60-h group was the highest (36.11 ± 1.78%) compared with the other groups (P < 0.05). Whereas the reconstructed embryos derived from donor cells treated with TSA for 24 h were continually cultured in TSA for different times (24, 48 and 60 h), the results showed that the blastocyst rate (37.39 ± 1.78%) in the 60-h group was significantly higher than that of the 24-h (25.48 ± 1.34%) group (P < 0.05). Finally, when the cloned embryos from different groups were respectively transferred to 40 natural oestrus recipients, no significant difference in terms of pregnancy rate among groups was found; however, a viable cloned calf was successfully obtained from TSA-treated donor cells and cloned embryo. Therefore, cloned embryos treated with optimized methods can develop to term.

Table 1.Pregnancy results established from embryos of different origins
165 THE EFFECTS OF L-CARNITINE AND LINOLEIC ACID ALBUMIN SUPPLEMENTATION ON THE DEVELOPMENT AND CRYOSURVIVAL OF BOVINE IN VITRO-MATURED/IN VITRO-FERTILIZED EMBRYOS IN IN VITRO CULTURE MEDIUM
Authors: Miyashita S. , Inaba Y. , Somfai T. , Geshi M. , Nagai T. , Koyama H. , Dochi O.
Published: December 2011

The objective of this study was to investigate the effects of the supplementation of a lipid metabolism inducer, L-carnitine (LC) and a membrane stabilizer, linoleic acid albumin (LAA), on the developmental competence and cryosurvival of bovine in vitro-matured/in vitro-fertilized embryos in in vitro culture medium. Cumulus–oocyte complexes collected from the ovaries of slaughtered cattle were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 AU mL–1 of FSH at 38.5°C in an atmosphere of 5% CO2 in air. After IVF (Day 0), presumptive zygotes were cultured in CRlaa containing 5% CS at 38.5°C in an atmosphere of 5% CO2, 5% O2 and 90% N2 for 9 days. The culture medium was supplemented with 0.6 mg mL–1 of LC (LC group; n = 180) or with 0.25 mg mL–1 of LAA (LAA group; n = 180) or with both LC and LAA (LC + LAA group; n = 180) or without LC and LAA (control; n = 178). The cleavage rates were recorded on Day 2 and the blastocyst formation rates were recorded on Day 7 to 9. Expanded blastocysts harvested on Day 7 and 8 (LAA group: n = 31; LC group: n = 29; LC + LAA group: n = 25; control group: n = 33) were used for freezing in modified PBS supplemented with 1.5 M ethylene glycol, 0.1 M sucrose and 20% CS. After thawing, they were cultured in TCM-199 supplemented with 20% FBS and 0.1 mM β-mercaptoethanol at 38.5°C under 5% CO2 in air for 72 h. The rates of re-expansion, hatching and formation of hatched blastocysts were determined at 24, 48 and 72 h after thawing, respectively. The rates of cleavage and blastocyst formation were expressed as mean ± s.e.m. and analysed by ANOVA. The post-thaw survival rates of frozen embryos were analysed by chi-square test. The cleavage rate in the control group (69.1 ± 2.5%) was significantly lower than that in the LAA (81.8 ± 3.8%) and LC + LAA groups (77.9 ± 1.4%) but did not differ from that in the LC group (73.8 ± 2.4%). The blastocyst formation rate in the control group (21.7 ± 2.8%) was significantly lower (P < 0.05) than those in the LAA and LC + LAA groups (33.5 ± 2.8% and 31.4 ± 2.4%, respectively), but it did not differ significantly from that of the LC group (32.1 ± 3.3%) despite a strong tendency (P = 0.06). There were no significant differences among the control, LC, LAA and the LC + LAA groups in post-thaw re-expansion rates (66.7, 75.9, 67.7 and 76.0%, respectively), hatching rates (48.5, 69.0, 58.1 and 64.0%, respectively) and rates of formation of hatched blastocysts (51.5, 62.1, 61.3 and 64.0%, respectively). These results indicate that the addition of LC and LAA to the medium for in vitro culture of in vitro-matured/in vitro-fertilized bovine embryos improved their ability to develop to the blastocyst stage; however, the effects on the freezing tolerance were not verified.
186 RELIABILITY OF HOECHST 33342 STAINING UNDER STANDARD EPIFLUORESCENCE MICROSCOPY FOR EVALUATION OF THE NUCLEAR STATUS OF LIVING DOG OOCYTES
Authors: Chebrout M. , Adenot P. G. , Reynaud K. , Chastant-Maillard S.
Published: December 2011

Hoechst 33342 staining detected by standard epifluorescence microscopy (Epi) is widely used for nuclear stage determination of oocytes in numerous species. Because it maintains cell viability, observed oocytes can be further processed for assisted reproduction. This sorting would be of great interest in the canine, where oocytes resuming meiosis in vitro remain scarce. But because of the cytoplasmic opacity of the canine oocyte, the accuracy of this technique is questionable. The objective of this study was to evaluate the accuracy of Epi by comparison to a reference technique (confocal microscopy; Conf). Oocytes were obtained from anestrus ovaries of 46 pubertal bitches. In vitro maturation was conducted with 20 oocytes/well in 500 μL M199 with 20% fetal calf serum in a humidified atmosphere of 5% CO2 at 39°C for 72 h. After Hoechst 33342 staining (20 min at RT), oocytes were observed without fixation under Epi (IX70, Olympus, France) with the excitation beam attenuated by 150-fold and emission detected by a high sensitivity camera (CCD, Olympus). The mean time for capture was 300 ms. For Conf, oocytes were fixed, stained with Ethidium Homodimer-2 (2 μM for 20 min at RT) and examined with an Ion-Argon laser (LSM 310, Carl Zeiss, Germany). For each oocyte, the nuclear stage was successively determined by Epi and by Conf. The results of both observations were compared by chi-square analysis. The optimal concentration of Hoechst dye was first determined on 401 oocytes (200 ng, 500 ng, 1 μg, or 2 μg mL–1). The 1 μg mL–1 was the lowest concentration allowing the identification of the nuclear stage by Epi in the largest proportion of living oocytes seen with DNA by Conf. At this dye concentration, Epi did not significantly impact degeneration or meiosis resumption rates: 33.3 and 22.4%, respectively for 183 oocytes submitted to Epi before Conf, compared to 37.4 and 13.6% for 147 oocytes observed only under Conf. Finally, the concordance of the nuclear stage diagnosis was tested on 379 oocytes observed individually with Epi and Conf successively. On Epi, 149 oocytes were seen without DNA. Observed on Conf, 37.5% of these were also seen without DNA, 59.7% were in the germinal vesicle stage and 2% were in metaphase. On Epi, 89 oocytes were considered as having resumed meiosis. On Conf, meiosis resumption was confirmed for 88.7% of them, but Epi failed to detect 31.4% of the oocytes having resumed meiosis among the 379 cultured oocytes. Although 80% of the metaphase II oocytes identified by Epi were confirmed to have reached this stage, 23% of metaphase II oocytes present in the culture dishes were missed. In conclusion, probably because of the high density in lipids of the cytoplasm, Hoechst staining detected by Epi for canine oocytes sorting after in vitro maturation leads to erroneously discarding a high proportion of oocytes.
19 CHANGES IN PLASMA STEROID CONCENTRATIONS DURING GESTATION IN COWS WITH SPONTANEOUS ABORTION OF SOMATIC CELL CLONED FETUSES
Authors: Hirako M. , Takahashi H. , Kimura K. , Adachi N. , Akagi S.
Published: December 2011

Cloning of mammals by nuclear transfer frequently results in gestational failure with a variety of abnormalities that are likely due to inappropriate epigenetic reprogramming. Monitoring the placental function during gestation is important to clarify the cause of abnormalities in cloned animals. Sex steroids are produced in the bovine placenta and their levels in maternal peripheral blood are a useful measure of placentation. The objective of this study was to investigate changes in plasma concentrations of sex steroids during gestation in cows aborting cloned fetuses. Donor cells for nuclear transfer were obtained from subculture of cumulus cells retrieved from ovarian follicles of a Japanese Black cow. Recipient oocytes were derived from ovaries obtained at an abattoir and matured in vitro. Metaphase II oocytes were enucleated and each fused with a donor cell by DC pulses. Nuclear-transferred oocytes were activated and cultured for 7 days. Embryos developed to the blastocyst stage were each transferred into the uterine horn ipsilateral to the ovary bearing the CL of 39 multiparous Japanese Black and Holstein crossbred cows at 7 to 8 days after the day of standing oestrus (day 0). Fourteen recipient cows were diagnosed pregnant on Day 40 by ultrasonography and 7 cows delivered at full term. The other seven miscarried on Day 66, 81, 85, 89, 97, 104 and 211. Blood was collected from these cows at least once a week following the pregnancy diagnosis. Progesterone, estrone, oestradiol-17β and estrone sulfate in the blood plasma were measured by RIA and were compared with those in pregnant AI cows. Statistical differences at stages of gestation were analysed with repeated-measures ANOVA. In all miscarried cows, progesterone concentrations were similar to those in AI cows until several days before abortion and then rapidly decreased to the basal level. Concentrations of all estrogens stayed low until abortion in six cows aborting by day 104, whereas estrone and oestradiol-17β started to increase around Day 80 and estrone sulfate gradually increased from around Day 50 and started to increase drastically around Day 80 in AI cows. In another cow aborting on Day 211, profiles of estrone and oestradiol-17β were similar to those in AI cows until around Day 150. Thereafter, concentrations of these estrogens gradually decreased to the basal levels by Day 160 and stayed low until abortion. In this cow, gradual increase in estrone sulfate during Day 50 to 80 was not observed, but the difference in the concentration was not statistically significant from AI cows. The following profile of estrone sulfate was similar to those in active estrogens. The fetus was still alive on day 160 and fetal death was confirmed on day 180 by ultrasonography. These results suggest the possibility that developmental or functional failure of placenta associated with steroid synthesis may be a cause of mid-term miscarriage of a cloned fetus.