bluuebunny/crossref_metadata_2025 huggingface dataset:
Fabrication and magnetic properties of hard/soft magnetostatically coupled FePt∕FeNi multilayer microwires
Authors: Torrejón J. , Badini-Confalonieri G. , Vázquez M.
Published: January 2008

A family of multilayer microwires with hard/soft biphase magnetic behavior is here introduced. The microwires consist of a Fe63Pt27Si10 hard magnetic nucleus and a Fe20Ni80 soft outer shell separated by an intermediate insulating Pyrex glass microtube. The precursor FePtSi glass-coated microwire is fabricated by quenching and drawing technique, and its L10 hard magnetic phase is grown by postannealing treatment technique. The polycrystalline FeNi soft magnetic outer shell has been deposited by electroplating. The analysis of the low-field hysteresis loops of the FeNi soft phase after premagnetizing until near magnetic saturation provides information about the magnetostatic coupling between phases. The FeNi magnetization curve is shifted toward positive field when the FePt remanent magnetization is positive and vice versa. A systematic analysis of the magnetostatic coupling and the corresponding bias field arising from uncompensated poles of the premagnetized FePt hard phase has been performed. The strength of the bias field is shown to increase with the reduction of thickness of the FeNi layer. These magnetostatically coupled biphase systems are thought to be of large potential interest as sensing elements in sensor devices.
Computation of eddy current losses induced by magnetic domain walls motion with Ck-spline functions
Authors: Lakhdari Zakaria , Adélaïde Luca , Makany Philippe
Published: January 2008

This article presents a computation of the nonlinear dynamics of magnetic domain wall motion in ferromagnetic material such as crystalline, like Fe–Si, in formulation H, with interactions between walls, and bulk magnetic induction. These terms have important contributions to ferromagnetic losses in high exciting magnetic fields and are usually neglected. The dynamic trajectories of magnetic domain walls are given as nonlinear coupled ordinary differential time equations. Our simulations use the Ck-spline approach, which allows many algebraic facilities in algorithms and in boundary conditions.
Crystal structure and low temperature physical properties of Ho2CoGa8 intermetallic antiferromagnet
Authors: Adriano C. , Mendonça-Ferreira L. , Bittar E. M. , Pagliuso P. G.
Published: January 2008

We have synthesized single crystalline samples of Ho2CoGa8 intermetallic compound using a Ga-flux method. This compound crystallizes with a tetragonal structure, space group P4∕mmm, and lattice parameters a=4.219(5)Å and c=10.99(2)Å. This structure is a bilayer version of the HoCoGa5 (1-1-5) which hosts a series of heavy-fermion superconductors and complex antiferromagnetic intermetallic systems. Measurements of magnetic susceptibility, heat capacity, and electrical resistivity revealed that Ho2CoGa8 is a metallic Curie–Weiss paramagnet at high temperature and presents an antiferromagnetic ordering below TN∼5K. The low temperature magnetic properties of this compound show the effects of tetragonal crystalline electrical field and the Ruderman–Kittel–Kasuya–Yosid interactions and the results presented here are compared with a broader description of the evolution of the low-T magnetic properties of structurally related series of rare-earth based tetragonal 2-1-8 and 1-1-5 compounds.
Studies of magnetic properties and giant magnetoimpedance effect in ultrathin magnetically soft amorphous microwires
Authors: Zhukova V. , Ipatov M. , Gonzalez J. , Blanco J. M. , Zhukov A.
Published: January 2008

We report novel results on studies of giant magnetoimpedance (GMI) effect and magnetic properties in ultrathin amorphous glass-coated microwires with nearly zero and negative magnetostriction constant and metallic nucleus diameter ranging within 6 and 12μm. Off-diagonal MI curve of Co67.1Fe3.8Ni1.4Si14.5B11.5Mo1.7 microwire exhibits asymmetrical shape with almost linear growth within the field range from −Hm to Hm associated with the anisotropy field. Co74B13Si11C2 microwire possesses hysteretic off-diagonal MI curve. Current annealing significantly changes off-diagonal MI curves, especially in the case of Co74B13Si11C2 microwire.
Large energy density enhancement in FePt films by microstructure refining
Authors: Yuan F. T. , Wei D. H. , Chang H. W. , Chen S. K. , Huang H. W. , Hsiao S. N. , Sun A. C. , Lee H. Y. , Yao Y. D.
Published: January 2008

The effect of microstructure refinement on magnetic properties in single-phase Ll0 Fe58Pt42 films was studied. Samples with thickness t of 100, 200, and 300nm were sputtering deposited on glass substrate with elevated temperatures Ta of 400 and 800°C. In the t=300 series of films, the order parameter of films with Ta=400 and 800°C were identical, about 0.663, but the average grain size is different by approximately eight times in diameter (from ∼75to∼610nm). Comparing to the film of Ta=800°C with large grains, significant enhancement in remanence of about 32% was obtained in the 400°C annealed film (∼880emu∕cm3) and the energy density was achieved an increment of about 72% (19.6MGOe), which exceeds the theoretical preditions. The reasonable explanations are summarized as the dimension confinement of interactions and the degradation of anisotropy.
Two-dimensional critical scaling behavior of Barkhausen avalanches (invited)
Authors: Shin Sung-Chul , Ryu Kwang-Su , Kim Dong-Hyun , Akinaga Hiro
Published: January 2008

We have investigated critical scaling behavior of Barkhausen avalanches of two-dimensional Co and MnAs films using time-resolved magneto-optical microscopy, enabling to image Barkhausen avalanches at criticality. A stochastic analysis of the fluctuating size of Barkhausen jumps from numerous repetitive experiments shows a power-law scaling behavior in both systems. Strikingly, it is found that the variation of scaling exponent in the power-law distribution is closely correlated with the variation of the domain-evolution patterns. This result is understood by the fact that both the scaling exponent and the domain-evolution patterns strongly depend on the relative contribution between the dipolar interaction and domain wall energies.
Millisecond switching in solid state electrochromic polymer devices fabricated from ionic self-assembled multilayers
Authors: Jain Vaibhav , Yochum Hank M. , Montazami Reza , Heflin James R.
Published: January 2008

The electrochromic switching times of solid state conducting polymer devices fabricated by the ionic self-assembled multilayer method has been investigated. The devices were composed of bilayers of poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) and poly(allylamine hydrochloride) on indium tin oxide substrates. Devices fabricated from 40 bilayer thick films have coloration and decolaration switching times of 31 and 6ms, respectively, with low applied voltage (1.4V) for an active area of 0.6cm2. The switching times have been shown to decrease with the active area of the electrochromic device suggesting that even faster electrochromic switching times are possible for devices with smaller areas.
Accessory allowing an optical cell of 6–10000μm path for liquids and gases to be filled in vacuum and used under pressure
Authors: Lunelli Bruno
Published: January 2008

An accessory for introducing liquid or gaseous samples without any contamination into an evacuated cell for optical spectrometry is described. The procedure for its use is similar to that of sample injection into a gas-liquid chromatograph, no glove box or glove bag being needed. Main benefits are the wide choice of size of the samples, their recoverability, safety, and a range of optical paths allowing to measure the absorbance of strongly absorbing liquids and gases, very dilute liquid solutions, and liquefied gases under moderate pressure. Short path spectra of gaseous water pressure standards consent quantification of water partial pressure measured in common gas cells.
Magnetic switching of ferromagnetic nanotubes
Authors: Sharif R. , Shamaila S. , Ma M. , Yao L. D. , Yu R. C. , Han X. F. , Khaleeq-ur-Rahman M.
Published: January 2008

The magnetic switching of ferromagnetic nanotubes as function of geometrical parameters has been investigated. The modes of magnetization reversal are observed to depend on the geometry of the nanotubes. Time dependent magnetization properties reveal that the nanotubes have strong magnetic viscosity effects. The values of magnetic viscosity coefficient (S) for different applied fields are high near the coercive field.
Resonant dissociative electron transfer of the presolvated electron to CCl4 in liquid: Direct observation and lifetime of the CCl4*− transition state
Authors: Wang C.-R. , Drew K. , Luo T. , Lu M.-J. , Lu Q.-B.
Published: January 2008

We report a pump-probe femtosecond transient absorption spectroscopic study on the electron transfer reaction of CCl4 in liquid ethanol. By direct observations of the presolvated electron and of the reaction transition state CCl4*−, this study provides direct evidence of the resonant dissociative electron transfer (RDET) of the presolvated electron to CCl4. Moreover, the lifetime of CCl4*− in ethanol is directly obtained from the decay kinetics and its measured value is found to be nearly identical to its gas-phase value. Hence, these results also imply that RDET can be an efficient process in an aqueous environment.
Infrared analysis of the bulk silicon-hydrogen bonds as an optimization tool for high-rate deposition of microcrystalline silicon solar cells
Authors: Smets A. H. M. , Matsui T. , Kondo M.
Published: January 2008

It is demonstrated that the signature of bulk hydrogen stretching modes in the infrared of microcrystalline silicon (μc-Si:H) deposited at high deposition rates can be used for solar cell optimization in the high pressure depletion regime. A relation between the performance of a p-i-n solar cell and the hydride stretching modes corresponding to hydrogenated crystalline grain boundaries is observed. These crystalline surfaces show postdeposition oxidation and the absence of these surfaces in the μc-Si:H matrix reflects device grade microcrystalline material.
Expression of vascular endothelial growth factor receptor types 1, 2 and 3 in placenta from pregnancies complicated by hypertensive disorders
Authors: Marini M. , Vichi D. , Toscano A. , Thyrion G. D. Zappoli , Parretti E. , Mello G. , Gheri G. , Pacini A. , Sgambati E.
Published: January 2008

The aim of the present study was to determine the expression of vascular endothelial growth factor (VEGF) family receptors (VEGFR) in placentas from pregnancies complicated by hypertensive disorders of different clinical severity. Placental tissue from women with gestational hypertension, pre-eclampsia, pre-eclampsia with haemolysis, elevated liver enzymes and low platelets (HELLP syndrome) and normotensive women, as a control group, was examined. Immunohistochemical techniques, reverse transcription–polymerase chain reaction and western blot were used to evaluate receptor expression. In cases with gestational hypertension, as well as in control cases, VEGFR-1 and VEGFR-3 immunoreactivity was detected in all placental components, whereas in placentas from the pre-eclampsia and pre-eclampsia with HELLP syndrome groups, VEGFR-1 and VEGFR-3 immunoreactivity was detected only in some portions of trophoblast and/or some vessels and/or clusters of stromal cells. In the control group, VEGFR-2 immunoreactivity was observed only in the vessels, whereas the hypertensive groups showed VEGF-2 immunoreactivity also in trophoblast and stromal cells. The mRNA levels of the three receptors in the group with gestational hypertension were higher with respect to those in the control group. Placentas from pregnancies with pre-eclampsia showed lowest mRNA expression levels, whereas placentas from women with pre-eclampsia plus HELLP syndrome showed higher mRNA expression levels with respect to the three other groups. Receptor protein levels were lower in pathological cases compared with levles in the control group. These findings demonstrate a dysregulation of placental expression of VEGF family receptors related to the degree of clinical severity of the hypertensive disorder.
Relationship between delayed embryonic development and metabolic factors and fat deposition in fruit bat Cynopterus sphinx
Authors: Banerjee Arnab , Meenakumari K.J. , Krishna Amitabh
Published: January 2008

The present study was undertaken in the fruit bat Cynopterus sphinx, which breeds twice in quick succession at Varanasi, India. Its gestation period varies significantly in the two successive pregnancies of the year owing to delayed embryonic development during the first (winter) pregnancy. The primary aim of the present study was to determine the role of metabolic factors in delayed embryonic development in the fruit bat C. sphinx. Variation in bodyweight, fat deposition, oxygen (O2) consumption rate, basal metabolic rate (BMR), body temperature (Tb) and hepatic succinate dehydrogenase (SDH) activity, along with circulating levels of thyroid hormones (tri-iodothyronine and thyroxine), were examined as metabolic factors during the two successive pregnancies in C. sphinx. The increase in bodyweight observed in November was due to accumulation of white adipose tissue in the posterior abdominal region. A significant decline in O2 consumption rate, BMR, Tb and SDH activity was found in early winter in November–December, which coincides closely with the period of fat accumulation and with the period of delayed embryonic development in C. sphinx. A significantly higher O2 consumption rate, BMR, Tb and SDH activity was noted during the second pregnancy in, when embryonic development was relatively faster. Thyroid hormone levels were high during the period of embryonic delay compared with levels during the remaining months. The results of the present study suggest that the delayed embryonic development in C. sphinx during early winter may be due to a low O2 consumption rate, BMR, Tb and SDH activity in November–December. The energy saved by suppressing embryonic development in this species may be advantageous for fat accumulation. Increased thyroid hormone levels during the early winter period might facilitate fat accumulation in C. sphinx.
Laparoscopic ovum pick-up followed by in vitro embryo production for the reproductive rescue of aged goats of high genetic value
Authors: Baldassarre H. , Rao K. M. , Neveu N. , Brochu E. , Begin I. , Behboodi E. , Hockley D. K.
Published: January 2008

The efficiency of laparoscopic ovum pick-up (LOPU) followed by in vitro embryo production (IVEP) in the propagation of aged goats with poor reproductive performance was evaluated in the present study. Follicular development was stimulated in donor goats with 80 mg follicle-stimulating hormone and 300 IU equine chorionic gonadotrophin administered 36 h before LOPU. In addition, goats were heat synchronised with intravaginal sponges containing 60 mg medroxyprogesterone acetate for 10 days and a luteolytic injection of 125 µg cloprostenol 36 h before sponge removal and LOPU. Following in vitro maturation (IVM), oocytes were fertilised in vitro with frozen–thawed semen produced using the egg yolk-free Bioxcell extender (IVM, L’Aigle, France). The average number of follicles aspirated (17.9 ± 8.0 per goat), oocytes recovered (15.7 ± 8.4 per goat) and cleavage after IVM/in vitro fertilisation followed by a short 24-h in vitro culture in modified synthetic oviduct fluid medium (72 ± 7%) were similar to results reported previously by our group and others in younger goats. A total of 296 embryos was transferred into 50 heat-synchronised recipients, of which 40 became pregnant (80%) and 38 progressed all the way to term, delivering 86 live kids. The present study indicates that LOPU-IVEP can be used successfully to extend the reproductive life of valuable goats that have acquired difficulties becoming pregnant by artificial insemination after multiple kiddings.
Current ethical issues in animal biotechnology
Authors: Thompson Paul B.
Published: January 2008

The present paper reviews the current status of opinion and debate regarding ethical issues in three broad categories of relevance to animal biotechnology. The first is scientific integrity, where the focus has been on scientific fraud and the integrity of the research process. The second concerns possible harms or risks to parties affected either directly by research (including animals themselves) or through the eventual commercialisation or development of products from animal biotechnology. The final category concerns a responsibility to serve as a guardian of the public interest with respect to application and development of technologies derived from new genetic sciences. It is plausible to see the scientific community as a whole having such a fiduciary obligation to the broader public in virtue of the technical complexity of the issues and owing to the public funding and institutional support for scientific research. The overall conclusion is that in the latter two categories especially, there is an urgent need for new participation in deliberative consideration of ethical issues by working scientists.
1 DIFFERENCES IN RESUMPTION OF OOCYTE MATURATION IN YOUNG AND OLD MARES
Authors: Campos-Chillon L. F. , Clay C. M. , Altermatt J. L. , Bouma G. J. , Carnevale E. M.
Published: January 2008

The decline in fertility of aged mares is linked with declining oocyte quality. Oocyte viability is dependent on the ability of oocytes to remain in meiotic arrest until the initiation of maturation. We hypothesize that aging is associated with quantitative and temporal differences in meiotic arrest and resumption in oocytes, ultimately resulting in a dissociation of oocyte and follicular maturation. The objectives of this study were to determine temporal differences in the mRNA content of amphiregulin and epiregulin in granulosa cells; PDE4 in cumulus and granulosa cells; and PDE3A, GPR3, GDF9, and BMP15 in oocytes during in vivo maturation in young (3–12 years) v. old (>20 years) mares. Oocytes and follicular cells were collected by transvaginal follicular aspiration. Maturation was induced in estrous mares with a follicle >30 mm by injection of 750 �g of recombinant equine LH. Aspirations were attempted at 0, 6, 9, and 12 h after LH administration. Six oocytes and follicular cell samples from each age group and time point were collected and stored immediately after aspiration. Total RNA was isolated from single denuded oocytes and lysed cumulus and granulosa cells. A fraction of the total lysate was used to determine cell numbers from the DNA copy number of the equine CG� subunit gene. DsRED RNA was added to each RNA isolate to serve as an exogenous standard. Quantitative RT-PCR was performed from cDNA with equine primer pairs. Copy numbers were calculated with an intra assay standard curve of plasmid containing the specific gene and corrected with the exogenous DsRED RNA and cell number. For each gene, mean mRNA copy numbers for time points and age groups were compared by ANOVA and Tukey's HSD test. Expression of PDE4D in cumulus cells was similar between young and old mares and time points. However, PDE4D peaked (P < 0.05) at 6 h in granulosa cells from young, but not old mares. Amphiregulin expression in granulosa cells of young mares peaked (P < 0.05) at 9 h and did not increase in the old mares. Epiregulin expression in granulosa cells peaked (P < 0.05) at 9 h and 6 h in young and old mares, respectively. The pattern of expression of PDE3A for oocytes of young and old mares was similar with an increase (P < 0.05) at 9 h. There was an interaction (P < 0.05) in the expression of GPR3 for age � time. Expression peaked at 9 and 6 h in young and old mares, respectively. Pattern of expression of GDF9 was similar between young and old mares except for a decrease (P < 0.05) in expression in old mares at 9 h. There was an interaction (P < 0.05) in the expression of BMP15 for age � time. Expression in young mares peaked at 9 h while that in old mares peaked at 6 h and decreased at 9 h. These results suggest that key gene expression patterns involved in oocyte and follicular maturation cascades are asynchronous for young versus old mares and could explain some aspects of the age-associated decline in fertility.
106 EXPRESSION PATTERNS OF PROTEIN KINASE A SUBUNITS DURING MEIOTIC MATURATION OF PORCINE OOCYTES
Authors: Kim J. S. , Cho Y. S. , Song B. S. , Jin X. L. , Lee K. K. , Koo D. B.
Published: January 2008

Many extracellular ligands activate adenylyl cyclase and then alter the intracellular concentrations of the second messenger, cAMP. High intracellular concentrations of cAMP activate cAMP-dependent protein kinase A (PKA), which regulates the meiotic arrest of oocytes at the germinal vesicle (GV) stage. In this study, we investigated the expression patterns of PKA subunits in porcine oocytes during meiotic maturation. Porcine oocytes were matured in the NCSU-23 medium supplemented with 10% (v/v) porcine follicular fluid, 10 ng mL–1 of EGF, 25 µm β-mercaptoethanol, 0.57 mm cysteine, 10 IU mL–1 of pregant mare serum gonadotropin, 10 IU mL–1 of hCG, 1 mm dbcAMP for 22 h and further cultured without dbcAMP and hormone for 22 h. All experiments were repeated more than 3 times. All data were analyzed by using Duncan's test of ANOVA by Statistical Analysis System (SAS Institute Inc., Cary, NC, USA). We found that the expression of type I and type II regulatory subunits (RI and RII) of PKA were higher in the GV stage than those of metaphase II stage porcine oocytes. In addition, their expressions were dynamically changed from the metaphase I to metaphase II stage. The expression levels of PKA regulatory subunits type I and type II were evaluated byWestern blot analysis by using specific antibodies such as anti-PKAc, anti-PKARIα, anti-PKARIIα, and anti-PKARIIβ in porcine GV and metaphase II oocytes (n = 25). To confirm the relationship between PKA and cell cycle regulators such as MPF or MAPK during meiosis, the expression of maturation-promoting factor or mitogen-activated protein kinase were examined by Western blot with anti-cdc2 or anti-ERK1/2. The expressions of the RI and RII subunits of PKA were decreased, whereas the expression of maturation-promoting factor (cdc2) or mitogen-activated protein kinase (ERK1/2) were increased in metaphase II stage oocytes. In conclusion, our findings indicate that the dynamic change of PKA subunits plays an important role not only in cAMP/PKA signaling pathway but also in the regulation of meiotic progression beyond the metaphase II stage in porcine oocytes.
11 EVALUATION OF A CONTINUING EDUCATION PROGRAM FOR VETERINARIANS: OUTCOME ASSESSMENT OF APPLIED REPRODUCTIVE TECHNIQUES IN BEEF PRODUCTION
Authors: Di Croce F. A. , Bergonzelli P. H. , Soraci A. L. , Schuenemann G. M.
Published: January 2008

Continuing education programs are an important tool to improve the service quality of veterinarians (Moore et al. 1996 JAVMA 209, 1086; Moore et al. 2004 JVME 31, 146). The effectiveness of a continuing education program (CEP) designed to enhance the flow of applied, scientific-based, reproductive techniques (in addition to nutrition, economy, and animal health) from educators (n = 21) to beef veterinarians (n = 400) was evaluated during a period of four years (2003 through 2007) in Argentina. Control of the estrous cycle, artificial insemination, pregnancy diagnosis, development of replacement heifers, record keeping systems, breeding soundness examination, and reproductive health practices were included in the analysis. The CEP included lectures and hands-on practice activities (100 h year–1). Participants were evaluated prior to the enrollment (CON) in the CEP to determine reproductive weakness areas. The outcome assessment from educators to veterinarians (contents of the program and benefits for participants) of applied reproductive techniques was evaluated through a test of knowledge and a survey following the IACET guideline at the end of each academic year (TRT). Data were analyzed using the MIXED procedure of SAS (SAS Institute, Inc., Cary, NC, USA) in a cohort study. Prior to the enrollment, control of the estrous cycle, semen evaluation, and record keeping systems were found to be the major areas of weakness in CON participants. Participants agreed with the information provided in the program and they indicated that the contents learned were relevant (97 � 3%) and applicable (92 � 4%). Furthermore, the CEP met their expectations (97 � 3%) and improved the quality of the service (94 � 3%) of TRT participants. Development of replacement heifers, control of the estrous cycle, semen evaluation, record keeping systems, and reproductive health problems were indicated as the most important tools (>80%; P < 0.05) to improve beef production. Additionally, results of this evaluation did reveal new areas for improvement such as open communication channels between educators and practitioners, data management, and communication skills of participants, all of which may be necessary to increase the acceptance and impact of applied reproductive techniques by beef producers. In conclusion, effective professional CEP improved the quality service of participating veterinarians, adding new services and implementing new management strategies. For the CEP to truly be considered successful, the program must also reach the ultimate end user, the beef producers. Therefore, a detailed assessment of the real acceptance and impact of these applied reproductive techniques by beef producers is needed to improve our message delivery from educators to practitioners and beef producers.
116 EFFECTS OF EARLY PREGNANCY DIAGNOSIS USING ULTRASONOGRAPHY ON THE SUBSEQUENT EMBRYO AND FETAL LOSS IN DAIRY COWS
Authors: Kim I. H. , Lee J. I. , Kim U. H. , Kang H. G.
Published: January 2008

Under on-farm condition, pregnancy diagnosis can be rapidly and accurately diagnosed using ultrasound. However, the risk of the incidence of embryo and fetal loss following early pregnancy diagnosis has not been fully determined. This study assessed, using ultrasonography, the incidence of embryo and fetal loss in dairy cows following early pregnancy diagnosis after artificial insemination. A positive pregnancy was the recognition of the vesicle, embryo, or fetus by ultrasonography (Sonoace 600 with 5.0- or 7.5-MHz linear array transducer; Medison Co., Ltd., Seoul, Korea). Pregnancies (n = 702) were divided into three groups according to the number of days pregnant diagnosed: early A group (27 to 40 days, n = 143), early B group (41 to 50 days, n = 172), or standard group (51 to 70 days, n = 387). Following a positive pregnancy diagnosis, embryo or fetal loss included all cows with observed abortions and also cows found open after the positive pregnancy diagnosis. Data were analyzed by chi-square test or Fisher's exact test using an SAS program (SAS Institute, Inc., Cary, NC, USA). The incidence rate of embryo or fetal loss within 7 days after pregnancy diagnosis was 1.4, 0.6, and 0.3% for the early A, early B, and standard groups, respectively (P > 0.05). The incidence rate of the embryo or fetal loss during 8 to 30 days after pregnancy diagnosis was not different (P > 0.05) among the early A (0%), early B (1.2%), and standard groups (1.0%). Furthermore, the cumulative incidence rate of the embryo or fetal loss before calving was not different (P > 0.05) among the early A (9.8%), early B (9.3%), and standard groups (5.9%). In conclusion, early pregnancy diagnosis using ultrasonography does not significantly increase the risk of embryo and fetal loss in dairy cows.
This work was supported by the research grant of the Chungbuk National University in 2007.
117 THE REGULATOR OF THE UTERINE TRPV6 GENE DURING PREGNANCY IN RODENTS
Authors: Lee B.-M. , Lee G.-S. , Jeung E.-B.
Published: January 2008

Transient receptor potential cation channel, subfamily V, member 6 (TRPV6) is an epithelial Ca2+ channel protein expressed in calcium-absorbing organs. Recently it was reported that TRPV6 is expressed in female reproductive organs. The present study was performed to investigate the expression and regulation of uterine and placental TRPV6 during gestation in rats. In the uterus, TRPV6 was moderately expressed at pregnancy day (P) 0 and decreased until P4. Its expression level then peaked at P5, implying that TRPV6 may be involved in embryo implantation. Around P13, uterine TRPV6 expression increased slightly a second time, and then gradually decreased until the end of pregnancy at P21. Tissue localization of TRPV6 expression was determined by immunohistochemistry methods. TRPV6 protein was detected in uterine endothelium layers and glands; however, it was not detected in uterine tissues attached to the placenta. In the placenta, TRPV6 mRNA levels increased in mid-gestation, and the tissue distribution of placental TRPV6 protein suggests that it may play a role in transferring calcium to the fetus during pregnancy. To investigate the pathway(s) mediating TRPV6 expression during pregnancy in rats and mice, steroid hormone antagonists ICI 182 780 (ICI) and RU 486 (RU) were injected prior to maximal TRPV6 expression, as anti-estrogen and anti-progesterone agents, respectively. Pregnant rats (P4 for uterine RNA preparation, P19 for placental RNA preparation) received subcutaneous injections of RU (2.5 mg per rat), ICI (0.5 mg per rat), or a combination of RU and ICI. In addition, ICR-mice at P9 were injected with RU (25 µg per mouse) and/or ICI (2 µg per mouse). We measured level of TRPV6 mRNA transcription using semi-quantitative RT-PCR and real-time PCR. Data were analyzed by nonparametric one-way analysis of variance using the Kruskal-Wallis test, followed by Dunnett's test for multiple comparisons to vehicle. In rats, TRPV6 expression was reduced by RU treatment, and in mice ICI treatment blocked TRPV6 expression. Taken together, these results suggest that progesterone in rats and estrogen in mice act to regulate TRPV6 expression in the uterus and placenta during pregnancy. In conclusion, TRPV6 may play a role in embryo implantation in the uterus and in calcium transport between embryo and mother in the placenta.
120 EFFECTS OF BOVINE PLASMA LIPOPROTEINS ON ACCUMULATION OF CYTOPLASMIC LIPID DROPLETS AND MITOCHONDRIAL MORPHOLOGY IN BOVINE EMBRYOS
Authors: Abe H. , Sata R. , Tsujii H. , Hoshi H.
Published: January 2008

The excessive accumulation of cytoplasmic lipid droplets (LD) may affect the embryo quality such as the sensitivity of embryos to chilling and freezing. Previous studies demonstrated that the presence of serum in embryo culture medium caused the abnormal accumulation of cytoplasmic LD. However, the mechanism and source of cytoplasmic LD accumulation in bovine embryos cultured in serum-containing medium is unclear. In this study, the effects of bovine plasma lipoproteins (BPL) on the accumulation of cytoplasmic LD were investigated in bovine embryos. Bovine oocytes were matured in IVMD101 serum-free medium and inseminated in BO-based medium. After insemination, embryos were cultured in IVMD101 supplemented BPL (Sigma) at various concentrations (60, 150, 300, 600 μg mL–1) or 5% calf serum (CS) in a humidified atmosphere of 5% CO2 in air at 38.5°C. The embryos of 2-, 8-cell, morula, and blastocyst stages were used for experiments. Embryos were fixed and stained with Sudan black B (SBB) for histochemical evaluation. Some embryos were prepared for transmission electron microscopy. The effect of media supplemented with BPL on in vitro development of bovine embryos is shown in Table 1. The proportion of embryos developing to morula and blastocyst stages decreased with increased levels of BPL (Table 1). Histochemical examinations demonstrated that embryos cultured in BPLcontaining media had numerous lipid droplets in their cytoplasm as observed in the embryos developed in CS-supplemented medium. The increase in number of LD was dose-dependent. Electron microscopic observations demonstrated that large LD were abundant in the cytoplasm of morulae and blastocysts cultured in medium supplemented with BPL (300 μg mL–1). Many elongated mitochondria were found in embryos developed in IVMD101 medium at the morula and blastocyst stages, whereas many of the mitochondria in the morulae and blastocysts developed in IVMD101 medium supplemented with BPL or CS were of an immature form such as spherical or ovoid shape. These results indicate that bovine embryos cultured in BPL-containing media abnormally accumulated LD in their cytoplasm, suggesting that the BPL directly affects the accumulation of cytoplasmic LD. Furthermore, the excessive accumulation of cytoplasmic LD in embryos may affect the mitochondrial development of bovine embryos.

Table 1. Effects of BPL on in vitro development of IVM/IVF bovine embryos

This work was parially supported by Grant-in-Aid for Scientific Research (17380164), on Priority Areas ‘Lifesurveyor’(19021006) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Special Coordination Funds for Promoting Science and Technology of Japan, and the Japan Livestock Technology Association.
122 EFFECTS OF EMBRYO SEX AND GLUCOSE OR FRUCTOSE IN CULTURE MEDIA ON BOVINE EMBRYO DEVELOPMENT
Authors: Barceló-Fimbres M. , Seidel Jr G. E.
Published: January 2008

The objective of the present study was to determine if glucose, fructose, or both, in culture medium affects female embryos differently from male embryos. Embryos of predetermined sex were produced by use of sexed semen. Blastocysts were produced in vitro in a factorial design from 1851 oocytes fertilized with 3 sperm types [non-sexed control, X-bearing, and Y-bearing (~95% accuracy)]. Semen from 3 bulls (A, B, and C) was used, with 2 replicates for each bull. Zygotes were cultured with 4 energy substrates (no-hexose control, glucose, fructose, and glucose + fructose). Chemically defined medium (CDM) plus 0.5% fatty acid-free BSA (De La Torre-Sanchez et al. 2006 Mol. Reprod. Dev. 18, 585–596) was the culture medium to which the energy substrates were added. For the first 2.5 d of culture, the medium contained 0.5 mm hexose, and for the last 4.5 d, it contained 2.0 mm hexose. Data were analyzed by ANOVA. There was no effect of hexose treatments on cleavage rates (P > 0.1; Table 1). However, blastocyst rates per oocyte were higher for the fructose treatment than for the control, glucose, or glucose + fructose (P < 0.05) groups. Glucose treatment resulted in retarded development compared with no hexose or fructose (P < 0.05). Analysis of a subset of data with only the fructose and glucose treatments resulted in an interaction of hexose (glucose and fructose) and X-bearing andY-bearing sperm for blastocysts per oocyte: fructose X-sperm 20.4%a; fructoseY-sperm 17.7%ab; glucose X-sperm 10.6%bc, and glucoseY-sperm 14.7%c (means without common superscripts differ; P < 0.05). Glucose was detrimental to female embryos, and fructose improved development to the blastocyst stage for both sexes. Lower cleavage rates were found after fertilization with X-bearing sperm (Table 1); however, blastocyst rates per oocyte were not different after fertilization with X- or Y-bearing sperm or non-sexed sperm (P > 0.1). There was no bull effect on rates of cleavage or blastocysts per oocyte (P > 0.1). In conclusion, fructose improved embryonic development of embryos of both sexes; glucose retarded development of, and was toxic to, female embryos. Sexed sperm resulted in similar cleavage and blastocyst production as non-sexed sperm, and female embryos had retarded embryonic development relative to male embryos.

Table 1. Main effect least-squares means for development of bovine embryos (±SE)
124 A THREE-DIMENSIONAL IN VITRO IMPLANTATION MODEL WITH NONHUMAN PRIMATE EMBRYOS AND EXTRACELLULAR MATRIX UNDER VARIOUS CULTURE CONDITIONS
Authors: Chang T. , Bondarenko G. I. , Durning M. , Vielhuber K. , Garthwaite M. A. , Golos T. G.
Published: January 2008

The need for blastocyst culture and post-implantation embryo research has emerged in the past few years. Our objective is to evaluate a novel in vitro model to study implantation and placenta formation in vitro with rhesus macaque embryos under various culture conditions. A novel nonhuman primate in vitro 3-D system can provide cues for implantation and interaction with the extracellular environment not available in 2-D planar models. Optimization of such a model can be tested with diverse culture environments. We developed and evaluated an in vitro 3-D implantation model utilizing IVF-derived, blastocyst-stage rhesus macaque embryos embedded in 3-D Matrigel droplets cultured with different feeder cells and media. Signs of implantation including enlargement of the embryo mass, invasion and proliferation of trophectoderm cell layers, cystic formation, and cellular outgrowths derived from the embryo were initiated within the first week post-embedding. Trophoblast structures with protrusion and branches growing from the surface of embryo implants were observed. Immunohistochemical staining for chorionic gonadotropin (CG) combined with immunoassays for CG and progesterone indicated differentiation of trophoblastic cell lineages. In addition, we found morphological factors, such as proliferation of embryonic and extraembryonic structures, as well as initiation of protrusions interacting with the extracellular matrix, to predict successful establishment of prolonged embryo development. We further evaluated effects of different types of feeder cells and media combinations, and found that a combination of BRL, Ishikawa cells, and human uterine fibroblasts, provided an optimized culture microenvironment to promote peri-implantation embryo development and hormone secretion including CG and progesterone. In conclusion, we have established a 3-D in vitro system modeling implantation initiation, and demonstrating the capability of the embryo to interact with the extracellular matrix. Further studies will facilitate the methodology of peri-implantation blastocyst culture and accelerate our understanding of nonhuman primate embryo development, with potential for insights into early pregnancy loss and related pathologies. The present study and future directions may be extended to provide retrospective views on blastocyst selection for embryo transfer in assisted reproductive technology.
This study was funded by NIH grants RR000167, RR21876, and HD053926.
126 EFFECT OF ASSISTED HATCHING ON THE SURVIVAL AND THE DEVELOPMENT OF BOVINE EMBRYOS PRODUCED IN VITRO AFTER CRYOPRESERVATION
Authors: Fukuhara J. , Takuma T. , Kasa S. , Imai K.
Published: January 2008

The aim of this work is to investigate the effect of assisted hatching (AH) by partial zona pellucida (ZP) dissection on the survival and the development of bovine IVP embryos after ultra-rapid vitrification and slow freezing. COC obtained from abattoir bovine ovaries were matured and fertilized in vitro, and then cultured in IVD101 (Research Institute for the Functional Peptides, Yamagata, Japan) at 38.5�C in 5% CO2, 5% O2, 90% N2. The treatment of AH was done on compacted morulae by partially dissecting ZP with a micromanipulator. As a control, non-treated embryos with intact ZP were used. For vitrification, the blastocysts at days 7 and 8 were placed into a vitrification solution (Dulbecco's PBS (D-PBS) supplemented with 20% glycerol, 20% ethylene glycol (EG), 0.3 m sucrose (SUC), 0.3 m xylose, and 3% polyethylene glycol) for 30 s after two-step equilibration. Then, they were immediately placed on a custom-made vitrification tool made of nylon fishing line with a small piece of iron attached to one end (V-tool), and immersed into liquid nitrogen (LN2). After cooling, the embryos on the V-tool were placed into frozen 0.25 mL straws filled with a diluting solution (D-PBS supplemented with 0.5 m SUC and 20% new born calf serum) using a magnet, and then they were preserved in LN2. For warming, the straws were immersed into 25�C water. The V-tool was then introduced into the column of diluting solution using a magnet. For freezing, the blastocysts at days 7 and 8 were frozen by the conventional procedure with 10% EG. For thawing, the straws were immersed into 30�C water. In this study, 120 embryos were vitrified and 128 embryos were frozen. Warmed and thawed embryos were washed more than two times, and cultured in TCM199 supplemented with 20% fetal bovine serum and 0.1 mm β-mercaptoethanol for 72 h for assessment of survivability and developmental capacity of post-thaw embryos. Data were analyzed with the chi-square test. The survival rates of vitrified embryos were the same with or without AH (81.1 and 82.0%, P > 0.05). The survival rates of frozen embryos were also the same with or without AH (76.3 and 66.7%, P > 0.05). The survival rates of vitrified embryos without AH was significantly higher than that of frozen embryos without AH (82.0 v. 66.7%, P < 0.05). The hatched rates of frozen embryos without AH were significantly lower than that of frozen embryos with AH and those of vitrified embryos with and without AH (43.5 v. 64.4%, 67.9 and 68.9%, P < 0.05). These results indicated that AH enhanced the development of frozen bovine IVP embryos and that our vitrification method using a V-tool did not require AH for development of embryos.
129 IN VITRO PRODUCTION OF EQUINE EMBRYOS FROM YOUNG AND OLD MARES BY INTRACYTOPLASMIC SPERM INJECTION
Authors: Herrera C. , Revora M. , Vivani L. , Miragaya M. H. , Quintans C. , Pasqualini R. S. , Losinno L.
Published: January 2008

High merit mares obtain their utmost productive value at the same time their reproductive soundness diminishes. The aim of our study was to compare the developmental competence of equine oocytes from young and old mares after intracytoplasmic sperm injection (ICSI) and in vitro culture. Ovaries from young and old mares were obtained from a pool of slaughterhouse animals that have been previously selected by overall good body condition, reproductive status, and age. Young mares were 3 to 8 years old, and old mares were more than 15 years old. The age of all mares was determined by teeth observation and reproductive status by ultrasonography. Oocytes were obtained from ovaries 1 h postmortem by individual dissection of follicles between 10 and 25 mm and scraping of the follicle wall with a bone curette. Recovered oocytes were matured in vitro for 24–30 h, and all oocytes with an intact cytoplasm and a visible polar body were subject to ICSI and cultured for 7.5 days in SOFm. The maturation rate, cleavage, and embryo development rate and mean number of blastomeres at 7.5 days of culture were compared between oocytes and embryos from young and old mares. Maturation, cleavage, and developmental rates were analyzed by Chi Square and Fisher exact test, whereas the mean number of blastomeres at 7.5 days of culture was compared by one way ANOVA and t-test. A total number of 54 oocytes from young mares and 37 oocytes from old mares were obtained. There were no significant differences between the maturation, cleavage, or embryo development rates between young (79.63, 56.41, and 18.18%) and old (91.89, 63.33, and 15.79%) mares. In addition, the mean number of cells on embryos from each group did not differ significantly (57.75 v. 81; young v. old). Our preliminary results show that similar in vitro rates are achieved when oocytes from young or old mares are matured and fertilized in vitro by ICSI. This does not correlate with the reproductive senescence in old mares and the result obtained with other reproductive techniques. Further studies will determine if pregnancies are equally achieved using in vitro produced embryos from both age groups.
136 ADDITION OF LOW DENSITY LIPOPROTEIN TO CHEMICALLY DEFINED PORCINE EMBRYO CULTURE MEDIUM CAN PARTIALLY REPLACE BOVINE SERUM ALBUMIN
Authors: Spate L. D. , Walker K. A. , McHughes C. E. , Prather R. S.
Published: January 2008

Embryo culture media typically contain undefined biologicals such as BSA. Our goal is to develop chemically defined culture media that are based on the biology and physiology of the embryo. To that end we evaluated the presence of message in embryos at various stages of development and determined that the message for the low density lipoprotein receptor (LDLR) increased from the germinal vesicle and 4-cell stage to the blastocyst stage of porcine embryogenesis. Thus, this study was conducted to determine if the addition of low density lipoprotein (LDL) would enhance the development and quality of in vitro produced porcine embryos in an already chemically defined culture medium. Slaughterhouse ovaries were aspirated, cumulous–oocyte complexes (COC) identified, and the COC were matured for 42 h in M199 base medium supplemented with EGF, FSH, and LH. Metaphase II oocytes were then selected. Fertilization was then preformed in modified Tris buffered medium and cocultured with 0.25 � 106/mL frozen thawed porcine semen for 5 h. The presumptive zygotes were then transferred to either porcine zygote medium with 0.3% BSA or 0.1% PVA (PZM3, PZM4). After 28 h, cleaved embryos were then sorted into six treatment groups (1. PZM3, 2. PZM3 + 20 µg mL–1 LDL, 3. PZM4, 4. PZM4 + 10 µg mL–1 LDL, 5. PZM4 + 20 µg mL–1 LDL, 6. PZM4 + 50 µg mL–1 LDL). The embryos were cultured in 5%O2 5%CO2 90%N until day 7. The percentage of development to the blastocyst stage was determined and analyzed with the SAS Proc GENMOD Procedure (a–cP ≤ 0.05). The percentage blastocyst was 51.3 � 0.09a, 51.6 � 0.09a, 33.1 � 0.99c, 35.8 � 0.09c, 36.9 � 0.09c, and 41.3 � 0.06b, for treatments 1–6, respectively. Culture in PZM4 (without BSA) significantly reduced development. However, addition of 50 µg mL–1 of LDL to PZM4 improved development above PZM4 alone. We interpret these data to indicate that a high concentration of LDL in the PZM4 media did improve embryo development and that LDL could partially substitute for BSA. Differential staining was performed on the blastocysts, and preliminary results suggest that the ICM to trophectoderm ratio in the high LDL treatment group is closer to the ratio found in in vivo produced embryos.
This project was supported by USDA CSREES NRI (2006-35203-17282) and Food for the 21st Century.