bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative x-ray imager (abstract)
Authors: Evans Scott C. , Archuleta Tom N. , Oertel John A. , Walsh Peter J.
Published: February 2004

We report on development of a quantitative x-ray imager (QXI) for the national Inertial Confinement Fusion Program. Included in this development is a study of photocathode response as a function of photon energy, 2–17.5 keV, which is related to diagnostic development on the National Ignition Facility (NIF). The QXI is defined as being a quantative imager due to the repeated characterization. This instrument is systematically checked out, electronically as well as its photocathode x-ray response, both on a direct current and pulsed x-ray sources, before and after its use on a shot campaign. The QXI is a gated x-ray imager1 used for a variety of experiments conducted in the Inertial Confinement Fusion and Radiation Physics Program. The camera was assembled in Los Alamos and has been under development since 1997 and has now become the workhorse framing camera by the program. The electronics were built by Grant Applied Physics of San Fransisco, CA.2 The QXI has been used at the LANL Trident, LLNL Nova, and University of Rochester Laboratory OMEGA laser facilities. The camera consists of a grated microchannel plate (MCP), a phosphor coated fiberoptic faceplate coupled to film for data readout, along with high speed electronic pulsers to drive the x-ray detector. The QXI has both a two-strip and a four-strip detection head and has the ability to individually bias the gain of each of the strips. The timing of the QXI was done at the Trident short pulse laboratory, using 211 nm light. Single strip jitter was looked at as well and determined to be <25 ps. Flatfielding of the photocathode across the MCP was done with the Trident main laser with 150 J on a gold disk with a 1 ns. Spatial resolution was determined to be <5 μm by using the same laser conditions as before and a backlit 1000 lp/in. grid. The QXI has been used on cylindrical implosion work at the Nova Laser Facility, and on direct-drive cylinder mix and indirect-drive high convergence implosion experiments at OMEGA. Its two-strip module has provided the capability to look at point backlighters, as part of technique development for experiments on the NIF. Its next use will be in March 2000 with its off axis viewer nose at Omega, providing a perpendicular view of Rayleigh–Taylor spike dissipation.
Millimeter-wave reflectometry for electron density profile and fluctuation measurements on NSTX
Authors: Kubota S. , Nguyen X. V. , Peebles W. A. , Zeng L. , Doyle E. J. , Roquemore A. L.
Published: February 2004

A millimeter-wave reflectometry system for electron density profile and fluctuation measurements is being developed and installed on the National Spherical Torus Experiment. The initial frequency coverage will be in the bands 12–18, 20–32, and 33–50 GHz, provided by frequency-tunable solid-state sources. These frequencies correspond to O-mode cutoff densities ranging from 1.8×1012 to 3.1×1013 cm−3, which will span both the plasma core (ρ=r/a<0.8) and edge (ρ>0.8) regions. Operated as a broadband swept-frequency (frequency-modulated continuous-wave) reflectometer, the diagnostic is expected to provide routine (shot-to-shot) time- (⩽50 μs) and spatially resolved (∼1 cm) density profiles. The previous hardware can be easily reconfigured as a fixed-frequency reflectometer for density fluctuation measurements. The combination of measurements would be valuable for studying phenomena such as possible L- to H-mode transitions and edge-localized modes.
Time of flight electron spectrometry on TIR using natural diamond detector
Authors: Krasilnikov A. V. , Makarov K. N. , Rastyagaev I. N. , Satov Yu. A. , Smakovskiy Yu. B. , Alekseyev A. G. , Amosov V. N.
Published: February 2004

Natural diamond detectors (NDDs) being developed for the spectrometric applications in tokamak fusion plasma experiments, have been used also for fast x-ray flux measurements and time-of-flight (TOF) electron spectrometry in laser beam-target interaction experiments on the CO2 laser installation TIR (λ=10.6 μm, E=80–95 J, τ∼14 ns). The magnetic field created in front of the detector provided separation of the signals induced by x ray and electrons. The waveform of a NDD current response has been shown to provide an information about energy spectra of the particles generated during laser beam-target interaction. Fast response time (<1 ns), low sensitivity to visible light and x rays, high radiation resistance, and 100% charge collection efficiency of the type IIa diamond samples, make NDDs to be the optimal choice for TOF spectrometry of charged fusion products in inertial confinement fusion experiments.
Development of a neutron imaging diagnostic for inertial confinement fusion experiments
Authors: Morgan G. L. , Berggren R. R. , Bradley P. A. , Cverna F. H. , Faulkner J. R. , Gobby P. L. , Oertel J. A. , Swenson F. J. , Tegtmeier J. A. , Walton R. B. , Wilke M. D. , Wilson D. C. , Disdier L.
Published: February 2004

Pinhole imaging of the neutron production in laser-driven inertial confinement fusion experiments can provide important information about the performance of various capsule designs. This requires the development of systems capable of spatial resolutions on the order of 5 μm or less for source strengths of 1015 and greater. We have initiated a program which will lead to the achievement of such a system to be employed at the National Ignition Facility (NIF) facility. Calculated neutron output distributions for various capsule designs will be presented to illustrate the information which can be gained from neutron imaging and to demonstrate the requirements for a useful system. We will describe the lines-of-sight available at NIF for neutron imaging and explain how these can be utilized to reach the required parameters for neutron imaging. We will describe initial development work to be carried out at the Omega facility and the path which will lead to systems to be implemented at NIF. Beginning this year, preliminary experiments will be aimed at achieving resolutions of 30–60 μm for direct-drive capsules with neutron outputs of about 1014. The main thrust of these experiments will be to understand issues related to the fabrication and alignment of small diameter pinhole systems as well as the problems associated with signal-to-background ratios at the image plane. Subsequent experiments at Omega will be described. These efforts will be aimed at achieving resolutions of about 10 μm. Proposed developments for new imaging systems as well as further refinement of pinhole techniques will be presented.
Neutron scattering study of the layered Ising magnet CsDy(MoO4)2
Authors: Khatsko E. N. , Zheludev A. , Tranquada J. M. , Klooster W. T. , Knigavko A. M. , Srivastava R. C.
Published: February 2004

The quasi-two-dimensional antiferromagnet CsDy(MoO4)2 is studied by neutron diffraction and quasielastic neutron scattering. The crystal structure of two low-temperature phases (below 120 K and below 40 K) is determined. An approximate structure of the magnetically ordered phase (TN=1.36 K) is proposed. In the ordered state the order-parameter critical exponent β=0.17(0.01), the in-plane correlation length exponent ν=0.94(0.07), and the staggered susceptibility critical index γ=1.01(0.04) were determined. Comparing these results to the exact solution for a 2D Ising magnet, we conclude that, although 2D behavior is apparent in CsDy(MoO4)2, there are deviations from the simple 2D Ising model.
Relaxation of temperature and concentration in superfluid He3–He4 mixtures. Effective thermal conductivity
Authors: Zadorozhko A. A. , Kalko T. V. , Rudavskii É. Ya. , Chagovets V. K. , Sheshin G. A. , Nemchenko K. É.
Published: February 2004

The relaxation of temperature and concentration of a superfluid He3–He4 mixture with an initial molar concentration of 9.8% He3 is investigated in the temperature interval 100–500 mK for different values of the heat flux. It is shown that the kinetics of the change in temperature of the liquid can be approximated by an exponential function, and the time constants obtained depend weakly on temperature. The concentration relaxation processes are analogous to the temperature relaxation processes only in the region of the single-phase mixture, while below the phase separation temperature the change in concentration with time is of a nonmonotonic character and can be described by a superposition of two exponential functions. This kind of kinetic behavior of the mixture is explained in the framework of a simple model which illustrates the distribution of the concentration over the height of the cell at various times after a heat flux is turned on. In this case the concentration relaxation of the dilute phase consists of two processes—growth of the concentration due to the participation of He3 in the normal motion, and decline of the concentration due to the formation and growth of the concentrated phase. The data on the relaxation times are used for determining the effective thermal conductivity of the mixture; the value obtained is in agreement with a calculation done in the framework of a kinetic theory of the phonon–impurity system of superfluid mixtures.
Kinetics of the phase transition in solid solutions of He4 in He3 at different degrees of supersaturation
Authors: Grigor’ev V. N. , Maidanov V. A. , Penzev A. A. , Rudavskii É. Ya. , Rybalko A. S. , Syrnikov Ye. V. , Slezov V. V.
Published: February 2004

Precision measurements of the pressure during phase separation in samples of solid solutions of He4 in He3 have been used to obtain data on the characteristic times of the phase transition. A processing of the results gives additional evidence supporting the view that homogeneous nucleation is realized in He3–He4 solid solutions at significant supercoolings and heterogeneous nucleation at the smallest supercoolings. Two different ways are proposed for comparing the results with a theoretical calculation taking into account the processes at the boundary of a nucleus of the new phase. Both give roughly similar values of the coefficient of surface tension at the nucleus–matrix boundary, and those values agree with those obtained in other studies. It is conjectured that the bcc–hcp transition has a substantial influence on the kinetics of separation at the lowest supersaturations.
Nonlinear conductance of a quantum contact containing single impurities
Authors: Avotina Ye. S. , Kolesnichenko Yu. A.
Published: February 2004

The conductance of a quantum contact containing single point defects and a potential barrier is investigated theoretically. The dependence of the conductance G on the applied voltage U is obtained for the model of a quantum wire connecting massive banks. The comparative values of the different nonlinear contributions to the conductance due to the interference of electron waves scattered by defects and by defects and the barrier are analyzed. The latter contribution becomes dominant even at extremely small coefficients of reflection of electrons from the barrier. It is shown that the dependence of the transmission coefficient T12 on the electron energy E explains the experimentally observed suppression of oscillation of the conductance G(U) when its absolute value is close to the single-quantum value G0=2e2/h.
Electron diffraction study of the structural transformations in free argon clusters
Authors: Danylchenko O. G. , Kovalenko S. I. , Samovarov V. N.
Published: February 2004

An electron diffraction technique is used to study the structure of clusters formed in an isentropically expanding supersonic argon jet. The formation of the hcp phase with increasing cluster size is reliably detected for the first time. Observations are made for mean cluster sizes N̄ in the range from 1×103 to 8×104 atoms/cluster. An analysis of the shape of the diffraction peaks is carried out. It is found that in the range N̄⩽2×103 atoms/cluster, where the clusters are icosahedral, the profiles of the diffraction peaks are well approximated by a Lorentzian. For fcc clusters with N̄⩾3×103 atoms/cluster a better approximation is the standard Gaussian function. In the case N̄⩾1×104 atoms/cluster one observes peaks of the hcp phase in addition to the fcc peaks. The intensity of the hcp peaks increases with increasing cluster size, and for N̄≈8×104 atoms/cluster, the (110), (101), (103), and (202) peaks, characteristic only for the hcp phase, are clearly registered in addition to the fcc peaks. A possible mechanism for the formation of the hcp structure in Ar clusters is proposed.
Developments in in vitro technologies for swine embryo production
Authors: Wheeler Matthew B. , Clark Sherrie G. , Beebe David J.
Published: February 2004

Several modifications have been made to in vitro production (IVP) systems to allow more efficient production of viable porcine embryos. Although in vitro production of pig embryos has been studied for over 30 years, the overall blastocyst production rate remains low. The low blastocyst rate is due to several factors, including polyspermic oocyte penetration, low rate of male pronucleus formation and less than optimal in vitro culture systems. These conditions are all inherent problems in porcine IVP and many of the mechanisms involved remain unknown. Considerable research has examined culture medium and the techniques used during the various stages of in vitro production. However, changes to the physical culture system used during IVF have remained unchanged until recently. The present paper will summarise selected developments in fertilisation and embryo culture media composition and focus on the development of modified equipment to improve the conditions used during the IVP of porcine oocytes and embryos.
Development of national animal genetic resource programs
Authors: Blackburn H. D.
Published: February 2004

Globally, animal genetic resources are contracting due to economic forces. As a result, during the 1990s there was a dramatic increase in national animal genetic resource activities. Many national programmes were initiated and the Food and Agriculture Organisation of the United Nations played a prominent role in coalescing national actions. Nationally, programmes have been initiated that comprise of in situ, ex situ and information-exchange efforts. A critical element to national conservation efforts is the development of cryopreserved collections of germplasm. Several countries have initiated substantial multispecies collections of cryopreserved germplasm. The selection of animals within breeds of interest is an important consideration in building cryopreserved collections. Animal selection should be based on a lack of genetic relationship, with sufficient numbers of animals to ensure the capture of rare alleles. Major issues facing repository development and genetic conservation are: (1) that all breeds are in need of genetic diversity management; (2) a better understanding of in situ breed population dynamics is needed; and (3) the concept that repository collections can be used by a broad range of clientele across time as well as during emergency situations.
An overview of genomics research and its impact on livestock reproduction
Authors: Rohrer Gary Alan
Published: February 2004

The amount of information currently available about the genomes of many livestock species, especially cattle and pigs, has increased dramatically in the past few years and the rate of its accumulation will continue to increase. A large number of scientific ‘tools’ is currently available for research aimed at understanding biological processes important to livestock production. These resources are being used to understand biological processes regulating reproduction, body composition and immune function in all livestock species. This research will create many opportunities to develop new tools for the improvement of livestock production. The best commercial application of genomics is in conjunction with reproductive enhancing technologies, such as artificial insemination, embryo transfer and cloning.
Serial analysis of gene expression (SAGE) during porcine embryo development
Authors: Blomberg Le Ann , Zuelke Kurt A.
Published: February 2004

Functional genomics provides a powerful means for delving into the molecular mechanisms involved in pre-implantation development of porcine embryos. High rates of embryonic mortality (30%), following either natural mating or artificial insemination, emphasise the need to improve the efficiency of reproduction in the pig. The poor success rate of live offspring from in vitro-manipulated pig embryos also hampers efforts to generate transgenic animals for biotechnology applications. Previous analysis of differential gene expression has demonstrated stage-specific gene expression for in vivo-derived embryos and altered gene expression for in vitro-derived embryos. However, the methods used to date examine relatively few genes simultaneously and, thus, provide an incomplete glimpse of the physiological role of these genes during embryogenesis. The present review will focus on two aspects of applying functional genomics research strategies for analysing the expression of genes during elongation of pig embryos between gestational day (D) 11 and D12. First, we compare and contrast current methodologies that are being used for gene discovery and expression analysis during pig embryo development. Second, we establish a paradigm for applying serial analysis of gene expression as a functional genomics tool to obtain preliminary information essential for discovering the physiological mechanisms by which distinct embryonic phenotypes are derived.
Integrating new technologies with embryology and animal production
Authors: Greve Torben , Callesen Henrik
Published: February 2004

The present review describes a range of selected farm animal embryo technologies used in embryological research and applied in animal breeding and production. Some of the techniques are driven by the breeder’s wish to obtain animals with higher breeding values, whereas others are primarily driven by the curiosity of researchers. The interaction between basic research and practical application in these areas is still a characteristic feature for people who contribute to the International Embryo Transfer Society (IETS) and has been an advantage for both researchers and breeders. One example of such an interaction is that detailed structural analyses have described quality differences between embryos of various origins and, following embryo transfer, the pregnancy results have confirmed the correlation between morphology and viability. Another example is that polymerase chain reaction technology has allowed detection of Y-specific sequences in male embryos and has become a tool in animal production today. Data from domestic animal genome sequencing will provide a great deal of new information. A major challenge for the years to come will be using this information in a physiologically meaningful context and to continue the efforts to convert the laboratory experience into use in practise. Finally, it is important to obtain societal acceptance for a wider application of many of the technologies, such as in vitro embryo production and cloning.
103A NEW PAPER CONTAINER FOR THE VITRIFICATION OF BOVINE EMBRYOS
Authors: Kim Y.M. , Ko D.H. , Uhm S.J. , Chung K.S. , Lee H.T.
Published: February 2004

Vitrification has been used to eliminate ice crystal formation during the cryopreservation of mammalian embryos. However, this method may introduce some problems such as loss of eggs during cryopreservation (EM grid) and damage to the zona pellucida. This study examined an alternative container (paper) for the vitrification of in vitro-produced bovine blastocysts. Bovine oocytes were aspirated from slaughterhouse ovaries and cultured in TCM-199 supplemented with 25mM NaHCO3, 10% (v:v) FBS, 0.22mM sodium pyruvate, 25mM gentamycin sulfate, 10μgmL−1 FSH (Follitropin V; Vetrepharm, Canada) and 1μgmL−1 estradiol-17β for 24h. Matured oocytes were co-cultured with sperm (1–106mL−1) treated by percoll gradient for 42–44h. Cleaved embryos were cultured in 50μL CR1aa medium containing 0.4% BSA for 5 days. Blastocysts were exposed to 5.5M ethylene glycol in CR1aa medium for 20s. The blastocyst suspensions were vitrified by one of three methods: 1) aspiration into a 0.25-mL plastic straw (10 embryos/straw), heat sealing and immediate plunging into LN2; 2) transfer of a (∼5μL) drop containing 10 blastocysts onto a EM grid and immediate plunging into LN2; or 3) transfer of a (∼5μL) drop containing 10 blastocysts onto a piece of weighing paper (5mm by 5mm; VWR, West Chester, PA, USA) and immediate plunging into LN2. Straws were thawed by holding in air for 10s and then transfer into 37°C water. The embryos were recovered from the straw and transferred into a solution of 0.5M sucrose in CR1aa at 25°C for 1min. EM grids and paper containers were warmed by transfer into 3mL of a solution of 0.5M sucrose in CR1aa medium at 25°C for 1min. Embryos were then diluted serially by transfer into 0.25 and then 0.125M sucrose solutions (1-min steps), and then rinsed and cultured in CR1aa medium supplemented with 10% FBS. After thawing, the recovery rates of embryos from EM grids, straws and paper containers were not significantly different (Table 1). Broken zonae pellucidae were observed after thawing of embryos recovered from straws and EM grids, but not from the paper container. The survival rates of blastocysts cryopreserved on EM grids and paper containers (respectively, 78.1 and 77.1%) were significantly higher (P<0.05) than that of straws (52.1%). The in vivo developmental potential of blastocysts vitrified on EM grids and paper containers was assessed by the transfer of, respectively, 102 and 3 thawed embryos into recipient cows. Pregnancy rates were, as anticipated, 28 and 67%. These results suggest that paper may be an inexpensive and useful container for the cryopreservation of mammalian embryos.

Table 1
The viability of vitrifield-thawed bovine embryos using various containers
104EFFECT OF HATCHING STATUS ON VITRIFICATION OF CLONED BOVINE BLASTOCYSTS
Authors: Laowtammathron C. , Terao T. , Lorthongpanich C. , Muenthaisong S. , Vetchayan T. , Hochi S. , Parnpai R.
Published: February 2004

Bovine blastocysts produced by nuclear transplantation have mechanical slits in their zonae pellucidae, and therefore initiate hatching earlier than the non-manipulated embryos. The present study was undertaken to examine whether the hatching stage of cloned blastocysts is among the factors influencing their survival after vitrification and warming. Cloned bovine blastocysts were produced by using adult ear fibroblast cells as reported previously (Parnpai et al., 2002, Theriogenology 57, 443), except that fused couplets were co-cultured with bovine oviductal epithelial cells in mSOFaa medium supplemented with 0.1% linoleic acid-albumin (LAA)+0.2% BSA (Hochi et al., 1999, Theriogenology 52, 497–504). Hatching blastocysts harvested on Day 7 were classified into one of three groups according to the ratio of extruding embryonic diameter from zona (D2) to embryonic diameter inside the zona (D1); category-A: D2/D1=0.01–0.70; category-B: D2/D1=0.71–1.00; category-C: D2/D1=1.01–1.70. The blastocysts were first exposed to 10% DMSO+10% ethylene glycol in TCM199+20% FCS for 2min, and then equilibrated in 20% DMSO+20% ethylene glycol+0.5M sucrose with or without 10% Ficoll in TCM199+20% FCS for 30s. One to three blastocysts were placed on a Cryotop sheet (Kitazato Supply Co., Tokyo, Japan) and vitrified in liquid nitrogen. The samples were warmed in 0.5M sucrose solution for 2min and transferred into TCM199+20% FCS in five steps (5min per step). The post-warm survival of the blastocysts was assessed by in vitro culture for 24h. When Ficoll-free vitrification solution was used, post-warm survival rate of the category-A blastocysts (77%, 23/30) was not significantly different (ANOVA test) from those of category-B and category-C blastocysts (74%, 20/27; and 80%, 24/30; respectively). Inclusion of 10% Ficoll in the vitrification solution did not improve (ANOVA test) the post-warm survival rates of cloned blastocysts (category-A: 65%, 22/34; category-B: 54%, 15/28; category-C: 59%, 19/32). Groups of fresh nonsurgical embryos, vitrified with or without Ficoll, yielded 66.7% (4/6), 66.7% (2/3) and 40.0% (2/5), respectively, of recipients pregnant at 48 days of gestation. In conclusion, cloned bovine blastocysts, regardless of their hatching stages, were relatively resistant to cryopreservation by vitrification. (Supported by Thailand Research Fund and R&D Fund of Suranaree University of Technology.)
108CRYOPRESERVATION OF GERMPLASM FROM HERITAGE BREEDS OF DOMESTIC
LIVESTOCK: SEASONAL EFFECTS ON SUPEROVULATION RESPONSE AND EMBRYO
PRODUCTION
Authors: Matsas D. , Huntress V. , Levine H. , Saperstein G. , Overstrom E.W.
Published: February 2004

There is an urgent need to preserve the genetic diversity of rare breeds of domestic livestock. However, it is unknown if conventional superovulation methods are effective in unimproved breeds. The establishment of germplasm cryostorage banks requires the ability to produce viable preimplantation stage embryos. To our knowledge, we describe here the first successful production and cryopreservation of embryos from Gulf Coast Native (GCN) sheep and Tennessee Myotonic (TM) goats. Mature GCN ewes and TM does were synchronized (prostaglandin F2α, 7.5–10mg i.m.) and all TM does, and some GCN ewes, were treated with progesterone (20mg sq, 5×on alternate days, prior to FSH treatment, then 10mg i.m. on second day of FSH treatment). Animals were superovulated (FSH, 50–40–30mg bid i.m., decreasing over 3 days) both in April (cycling) and June (anestrous). Females were bred by natural service with a proven male over 24hr, and embryos were collected surgically from the uterus on Day 7. FSH-stimulated cycling TM does produced a total of 15.3±3.0CL and 10.7±7.2 morulae/animal, whereas FSH-treated anestrous TM does yielded 9.0±2.8CL and 7.0±8.4 embryos per animal. Recovered goat embryos were morphologically of good-excellent quality, and were cryopreserved using a conventional multi-step freezing protocol. Cycling GCN ewes responded to FSH simulation and produced 9.7±2.5CL per animal, whereas the ovaries of non-cycling ewes did not respond to exogenous FSH treatment (0CL). This report documents that TM does will effectively respond to FSH superovulation treatment and produce viable embryos during the breeding season and during the anestrous period. In contrast, GCN ewes appear to respond only to exogenous FSH during the breeding season. Collectively, these results demonstrate that the TM and GCN landrace breeds of goats and sheep display differential responses to hormone manipulation; yet, with breed-optimized protocols, preimplantation stage embryos can be obtained for long-term cryostorage of germplasm of heritage breeds of livestock. Supported by and in partnership with SVF Foundation.
113COMPARISON OF TWO ETHYLENE GLYCOL EQUILIBRATION TREATMENTS FOR THE
QUICK FREEZING OF IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Nicácio A.C. , Simões R. , Yamada C. , Caetano H.V.A. , Mello M.R.B. , Assumpção M.E.O.A. , Gerger R.P.C. , Oliveira V.P. , Visintin J.A.
Published: February 2004

The aim of this study was to compare two ethylene glycol (EG) equilibration procedures for the quick freezing of in vitro-produced bovine embryos. Cumulus-oocyte complexes (COCs) were collected from slaughterhouse ovaries. COCs were matured in TCM199 containing 10% of bovine fetal serum, LH, FSH and E2, and fertilized. Presumptive zygotes were co-cultured in TCM199 with a granulosa cell monolayer, at 39°C in humidified atmosphere of 5% CO2 in air. Grade 1, expanded blastocysts (n=761) were selected 7 and 9 days after insemination and randomly distributed to one of eight treatment groups. In Equilibration Procedure 1, embryos were exposed to 10% EG for 5 min, and then to 17%, 22% or 28% EG for 60s (respectively referred to as EG 17, EG 22 and EG 28). In Equilibration Procedure 2, embryos were exposed to the same EG solutions as in Equilibration Procedure 1, but the period of exposure was 10min to 10% EG and 30 s to EG 17, EG 22 and EG 28. In Equilibration Procedure 3 (slow-freezing controls), embryos were exposed to 10% EG for either 5 or 10min and then cryopreserved by slow-freezing method at 1.2°C/min. In all treatment groups, EG solutions were prepared in PBS+0.2% BSA, and embryos were exposed to EG solutions at 22°C. Embryos were loaded into 0.25mL straws and heat-sealed. Straws were cooled in liquid nitrogen vapor for 2min, and then plunged into and stored in liquid nitrogen. Straws were thawed in room temperature air for 10s, and then in 25°C water for 20s. Thawed embryos were diluted by transferring them into 0.5ml of PBS+0.2% BSA+0.3M sucrose for 3min, and then 0.5mL of PBS+0.2% BSA for 3min. Embryos were co-cultured on granulosa cell monolayer in TCM199 and evaluated after 24h for blastocyst re-expansion (EXP), and again at 48, 72 and 96h for hatching (HAT). A total of 724 in vitro-produced bovine blastocysts were used as controls to determine hatching rates. The results are presented in the table. Embryos exposed to 10% EG for 10min (Equilibration Procedure 1) yielded significantly higher rates of blastocyst re-expansion and hatching when compared to embryos exposed for 5min (Equilibration Procedure 2, P<0.05). These results suggest that quick freezing of in vitro-derived bovine embryos may be an alternative to vitrification; however, additional studies are needed to optimize cryopreservation protocols and increase post-thaw survival. This project was supported by FAPESP (01/11266-4)

Table 1
Effect of equilibration procedure on in vitro re-expansion and hatching rates of embryos cryopreserved by slow and quick freezing methods
118VITRIFICATION OF PRONUCLEAR-STAGE MOUSE EMBRYOS ON ALUMINUM FOIL
FLOATING ON LIQUID NITROGEN
Authors: Sagirkaya H. , Ergin F. , Bagis H. , Arat S.
Published: February 2004

The cryopreservation of pronuclear-stage embryos has a special importance in transgenic technology, cloning, and human-assisted reproductive technology. The objective of this study was to investigate the efficiency of a vitrification method modified in our lab for pronuclear-stage embryos. In experiment I, groups of 10 pronuclear-stage mouse embryos were cultured in 20μL drops of three different culture media (G1.3/G2.3, CZB and M16) covered with mineral oil (Sigma M-8410, St. Louis, MO, USA). Twenty-four hours later, embryos cultured in G1.3 were transferred into G2.3 medium. In experiment II, 25–30 pronuclear-stage embryos were transferred into a 50-μL drop of equilibration medium containing 4% ethylene glycol (EG, Sigma E-9129) in TCM-199 (Sigma M-2520) supplemented with 10% FCS at 37°C for 12–15min; then they were rinsed three times in 30-μL drops of vitrification medium containing 35% EG, 5% polyvinylpyrrolidone (PVP, Sigma P-0930) and 0.4M trehalose (Sigma T-0167) in TCM-199 supplemented with 10% FCS at 37°C for 20–30s. Embryos rinsed in vitrification solution were aspirated into a micropipette as a 1–2-μL drop containing 25–30 embryos and dropped onto aluminum foil floating on liquid nitrogen (LN2). Vitrified droplets were stored in cryovials in LN2. Warming was performed by moving the vitrified droplets into 0.3M trehalose in TCM-199 supplemented with 10% FCS at 37°C. Embryos having normal morphological appearance under stereomicroscope examination were cultured in G1.3/G2.3 medium. Differences in the two experiments were analyzed by one-way ANOVA. In experiment I, development rates to the blastocyst stage were 26%, 10% and 4% for G1.3/G2.3, CZB and M16 media, respectively. The highest development rate in experiment I was obtained in G1.3/G2.3 culture media (P<0.05). Therefore, G1.3/G2.3 media were used for culturing of vitrified-warmed and control embryos. In experiment II, the rate of embryos having normal morphology was 98.5%. There were no significant differences between the development rates of vitrified (13.1%) and control (18.7%) embryos to the blastocyst stage (P>0.05). Although the vitrification method resulted in a high survival rate based on the morphological appearance, developmental rates of vitrified and control embryos were found to be lower than expected and reported previously by other researchers. We believe that the low developmental rates in this study were due to our culture conditions but not our vitrification method. Therefore, it could be concluded that this vitrification method is an efficient one for pronuclear-stage embryo cryopreservation and better development rates could be obtained by improving the culture conditions. This study was supported by a grant from TUBITAK, Turkey (VHAG-1908-102V048). F. Ergin is a young volunteer researcher.
121VITRIFICATION OF MOUSE MORULAE BY A NEW METHOD: PULLULAN FILM-STRAW VITRIFICATION
Authors: Takagi Y. , Shimizu M. , Kato T. , Danguri A. , Sakamoto M.
Published: February 2004

Recent technical improvements have resulted in higher cryosurvival of oocytes and embryos of various species. However, almost all methods require thawing and washing the embryos under microscopic observation and, therefore, cannot be conveniently used for large animal ET in the field. The purpose of the present work was to develop a new embryo cryopreservation method using a water-soluble film made of pullulan (Hayashibara, Okayama, Japan) that might, in the future, be readily adaptable to field conditions. Morula-stage mouse embryos were collected from superovulated ICR donors 72h after hCG injection. Embryos were first exposed to 10% DMSO+10% ethylene glycol (EG) in DPBS+20% FCS (mPBS) for 2min, and then equilibrated for 30s in a vitrification solution composed of 20% DMSO+20% EG+0.6M sucrose in mPBS. In the pullulan film-straw vitrification method, the embryos were loaded onto the pullulan film (20μm thick, 5mm long and 1mm wide) and were directly plunged into LN2. The pullulan film was inserted into a pre-frozen 0.25mL plastic straw (<−150°C) containing 0.15mL mPBS and sealed with a plastic screw cap. For thawing, the medium in the straw was rapidly warmed in 37°C water while the pullulan film remained frozen by placing the top of the straw in contact with a cold iron block (<−150°C). As soon as the medium thawed, the pullulan film was immersed in the medium by a rapid downward swinging of the straw. Five min later, embryos were recovered from the straw and washed for 2min in mPBS, for 2min in 0.1% BSA-PBS and for 2min in KSOM sequentially, and then cultured at 37°C in 5% CO2 for 38h. Noncryopreserved embryos and embryos cryopreserved by the cryoloop method (Lane et al., 1999 Nat. Biotech. 17, 1234) served as controls. Data were analyzed by χ2 test and Student’s t-test. Results are shown in Table 1. There are no significant differences (P>0.05) in either developmental abilities or cell numbers between vitrified and non-vitrified embryos. This study demonstrates that mouse morulae can be successfully vitrified and thawed by the PFSV method. This method may eventually be applied to bovine ET under field conditions.

Table 1
Development of mouse morulae in culture following vitrification by the PFSV method
133SUBOPTIMAL OOCYTE ACTIVATION CAUSES METHYLATION CHANGE IN TWO
IMPRINTED GENES IN THE MOUSE
Authors: Toth S. , Imamura T. , Kerjean A. , Heams T. , Madoux S. , Huneau D. , Ozil J.-P. , Paldi A.
Published: February 2004

In mammals, the dynamic of Ca2+ oscillations induced by fertilization causes the remodeling of the parental chromosomes and activates the developmental processes. The objective of the present study was to evaluate whether the modulation of the regime of Ca2+ oscillations during the process of egg activation is capable of inducing specific chromatin modification. The sensitivity of chromatin modification during oocyte activation was evaluated by comparison of the methylation profile of Igf2r, the maternal allele of which is methylated, and H19, the maternal allele of which is unmethylated. Freshly ovulated mouse oocytes were parthenogenetically activated by a series of repetitive Ca2+ ion influxes induced by electropermeabilization of the plasma membrane in a microfluidic processor. The first treatment (T1) consisted of 24 electrical pulses (1.45kVcm−1) given every 8min for 3h in the presence of 100μM of Ca2+. The treated oocytes were almost all activated, 98% (328/338), and formed pronuclei 3 or 4h after the first pulse. The second treatment (T2) was made up of 16 electrical pulses of lower amplitude (1.12kVcm−1), given every 8 min for 2h under the same conditions. The rate of egg activation dropped to 29%, (86/298), and the time course of pronucleus formation was completed 7h after the first pulse. The remaining oocytes, 71% (212/298), extruded the second polar body, but their chromatin did not undergo full decondensation and then did not proceed further to pronucleus formation. The methylation profiles of the two imprinted genes were analyzed by using the bisulfite sequencing method. Oocytes that formed pronuclei after being subjected to either the T1 or the T2 treatment displayed methylation profiles of the two alleles similar to those of the non-treated oocytes. The Igf2r-DMR was found fully methylated on all 13 copies examined, and the H19 DMR was found mostly unmethylated (13 out of 19 copies; 6 were partially methylated). Thus, when parthenogenetic activation results in formation of a pronucleus the methylation profile of these two genes is maintained. In contrast, those from oocytes that responded by PB extrusion showed the following: fully unmethylated Igf2r-DMR copies (9/9) and partially methylated H19-DMR copies (4/11). These results reveal that rapid de novo methylating and demethylating activity are present when cells are subjected to partial activation. Thus, when the biological response of the oocyte is incomplete, that is to say, when the chromatin remains partially decondensed after suboptimal activation, both active demethylation and de novo methylation can be remarkably dynamic. While the molecular mechanism needs to be clarified, these results reveal that modulation of the process of egg activation might give new opportunities to better understand the epigenetic chromatin changes caused by fertilization.
138EARLY EMBRYO MORTALITY IN BUFFALO
(BUBALUS BUBALIS) DURING THE SPRING PERIOD
Authors: Neglia G. , Grassi C. , Prandi A. , Galiero G. , De Carlo E. , Zicarelli L. , Campanile G.
Published: February 2004

Embryo development is linked to several factors, such as intrinsic defects within the embryo, an inadequate maternal environment or failure of maternal response to embryonic signals. Reproductive seasonality in buffalo species (Bubalus bubalis) is responsible for the lower reliability of the pregnancy diagnosis by progesterone assay in spring, probably due to the high incidence of early embryo mortality (EEM) for hormonal reasons (Campanile et al., 1989; Proc. 2th National Meeting ‘Studio dell’efficienza riproduttiva degli animali di interesse zootecnico’: 51–57). The aim of the study was to identify the causes of EEM in buffaloes inseminated during the spring period. Italian Mediterranean Buffalo cows (n=209) in a good state of health were synchronized using the Ovsynch-TAI Program (Pursley et al., 1995 Theriogenology 44, 915–923) and inseminated 16 and 40 hours after the second administration of GnRH. Artificial inseminations were performed by the same technician. Ten to 20 days after the first insemination two blood samples were collected from each animal for assessing P4 levels by RIA. Days 24 and 40 after the first insemination, pregnancy diagnosis was assessed by rectal ultrasonography (Aloka SSD-500) with a 5MHz sector scanner. In animals with EEM a uterine flushing was carried out and recovered fluid was analyzed for the principal infectious agents. Microbiological analyses were carried out according to Quinn et al. (1994 Clinical Veterinary Microbiology, Wolfe Publishing, Mosby, Europe) and a PCR analysis was performed only for Bubaline Herpes Virus (BuHV1), Toxoplasma and Neospora. Statistical analysis was assessed by Student’s t-test. The incidence of cyclic buffaloes after synchronization treatment (94%) and pregnancy rate at 40 days (34.4%) were similar to those reported in a previous study (Neglia et al., 2003 Theriogenology 60, 125–133). EEM between 24 and 40 days was 45% and only in 9 buffaloes (15.3%), these results correlated with the presence of infectious agents (Streptococcus spp. group B and G, Bacillus licheniformis, Proteus mirabilis, Staphylococcus spp.). P4 levels on Day 10 were higher (P<0.05) in pregnant (P) buffaloes than in those with EEM. On Day 20, P4 levels were higher (P<0.01) in P buffaloes than in those with EEM and those not pregnant. It is hypothesized that the reduced activity of hypophysis, correlated with an increase in hours of light, may be responsible for decreased P4 concentrations and EEM in buffalo.

Table 1
P4 blood levels in pregnant (P) and non pregnant (NP) buffaloes and in buffaloes with EEM 10 and 20 days after insemination
141EFFECT OF NUMBER OF LAMBS, THEIR SEX AND BIRTH WEIGHT ON OVINE
PREGNANCY-ASSOCIATED GLYCOPROTEIN (ovPAG) CONCENTRATIONS
Authors: Vandaele L. , Verberckmoes S. , De Cat S. , El Amiri B. , Sulon J. , Duchateau L. , Van Soom A. , Beckers J.-F.
Published: February 2004

Early pregnancy detection and prediction of the number of lambs would be profitable for sheep breeders, as this enables nourishment of pregnant ewes to their individual needs. Nonpregnant ewes can be separated and sold before winter. Prediction of sex is interesting for the curiosity of impatient breeders. Early pregnancy detection (before 45 days after mating) by means of ovine pregnancy-associated glycoproteins (ovPAGs) has proved to be a more reliable test than the commonly used transrectal ultrasound. The concentration of ovPAG has been claimed to be related to the number of lambs, their sex and birth weight, but contradictory results were obtained in different small-scale studies. In this study the correlation between number of lambs, their sex and birth weight and the ovPAG concentration in the serum of the ewes was examined. In a field trial, 160 and 70 ewes of the Texel and Suffolk breed, respectively, housed on 6 different farms, were synchronized by means of intravaginal sponges impregnated with 40mg cronolone (Chronogest®, Intervet, Boxmeer, The Netherlands). At the moment of synchronisation (T0) and at 25 (T25), 35 (T35) and 45 (T45) days after mating or artificial insemination, blood samples were taken by jugular vein puncture. A heterologous radioimmunoassay (RIA) was used to determine the ovPAG concentration. In the heterologous RIA boPAG molecules were used as tracer, and ovPAG molecules as standard, while rabbit antibodies against ovPAG were used as antiserum. At parturition, number, sex and birth weight of the lambs were registered and their effect on ovPAG concentrations was statistically evaluated. No significant difference in ovPAG concentration was found between ewes pregnant of a single male or female lamb (P=0.29). ovPAG concentrations were affected by the total birth weight (P<0.05). In ewes pregnant with multiples, the ovPAG concentrations were higher (P<0.01) than in ewes pregnant with singles at T25, T35 and T45, respectively. Because ovPAG concentrations were affected by the farm, it was impossible to predict the number of lambs by the ovPAG level. In conclusion, ovPAG concentrations were not affected by the sex of the lamb. However, ovPAG concentrations were related to the total birth weight. Within the farm, increased ovPAG concentrations may indicate pregnancy of multiples.
155OXYGEN MEASUREMENTS AS INDICATORS OF CULTURE CONDITIONS AND
VIABILITY OF CATTLE EMBRYOS
Authors: Lopes A.S. , Ottosen L.D.M. , Greve T. , Callesen H.
Published: February 2004

Experiments were performed to study oxygen and temperature conditions experienced by embryos during routine in vitro production (IVP) (Exp. 1) and to evaluate oxygen consumption of single embryos as a viability indicator (Exp. 2). In both experiments, bovine IVP embryos were used (Holm et al. 1999, Theriogenology 52: 683–700), and a combined oxygen-temperature microsensor (tip diameter approx. 50μm) was used to attain oxygen partial pressure gradients and temperature profiles. In Exp. 1, 4-well dishes with in vitro culture (IVC) medium (400-μL or 50-μL droplets) covered with 400μL oil were taken from the incubator (5% CO2 , 5% O2 , 90% N) and measured under atmospheric air conditions at room temperature (24–25°C). The profiles were determined over 2min, starting 2min after the dishes were taken out of the incubator. The 400μL medium was measured from the top layer and gradually 3/4 down into the well (seven replicates). The oxygen partial pressure decreased from 21.0±0.66% O2 at the top layer to 15.0±1.6% O2 at the bottom layer, while the average temperature increased from 27.2±3.0°C to 31.7±0.7°C. In the 50-μL droplets, the profiles were obtained in the middle of the droplet (five replicates). The oxygen partial pressure was 17.1±2.44% O2 and the temperature 31.0±1.11°C. Consequently, routine handling of culture dishes outside the incubator seems to have a strong influence on both temperature and oxygen conditions. In Exp. 2, Day 3 and Day 7 embryos were evaluated morphologically before being loaded consecutively into one of the holes in a measuring block developed for this purpose. The block was previously covered with 40mL IVC medium before being incubated for 3 days under 5% CO2 , 95% N (38.5°C, 100% humidity). The oxygen consumption of each embryo was measured from 75±5min after removal from the incubator. Each measurement lasted approx. 5min, was repeated three times for each embryo, and was performed under atmospheric conditions on a warming stage held at 37±1°C. The average oxygen consumption by Day 3 embryos was 0.25±0.14nL/embryo/h (n=20), and no clear relation between respiration rate and embryo morphology could be demonstrated. For Day 7 embryos, the average oxygen consumption was 0.90±0.56nL/embryo/h (n=22), and this varied according to their morphological quality (1.17±0.70 (n=7), 0.98±0.49 (n=6) and 0.46±0.38 (n=9) nL/embryo/h for good, fair and poor quality embryos, respectively). In conclusion, this study illustrated the changes in O2 partial pressure that embryos can temporarily be exposed to under routine handling, even for rapid procedures such as cleavage inspection. The results also show that respiration rate is lower in Day 3 than in Day 7 embryos. Furthermore, oxygen consumption values of Day 7 embryos seem to be in agreement with the morphological embryo quality, supporting the idea that oxygen consumption can be a valuable parameter for the evaluation of embryo viability.