A comparison of culture and cooling for the short term preservation of in vivo derived dromedary camel embryos of varying morphological quality.

Despite recent advancements in the cryopreservation of dromedary camel embryos, widespread application of the technique is still limited by the need for specialised vitrification equipment and supplies. Temporary, liquid-phase embryo storage methods provide a useful tool for short-term preservation of camel embryos. In the current study, we compared the use of in vitro embryo culture with cold liquid storage in order to maintain both high- (Grade 1- Excellent and 2-Good) and low- (Grade 3- Moderate and 4-Poor) morphological grade Day-7 dromedary camel embryos in vitro for up to 3 days. Embryos were either cooled and placed in Hams-F10 medium supplemented with HEPES and 10% FBS and then kept at 4 °C; or placed in Hams-F10 supplemented with sodium bicarbonate and 10% FBS and then cultured in a humidified atmosphere of 6% CO2 at 37 °C before being assessed for viability at 24 h. In high-morphological grade embryos, both cold storage and culture supported 100% viability (maintenance of normal morphology) over this period (Cooled n = 22, Cultured n = 20). In low-morphological grade embryos, culture supported higher viability (16/18, 88.9%) than did cooling (4/18, 22.2%). We then evaluated the effect of up to 3 days of cold storage or culture on embryo morphological grade, diameter, and developmental competence following embryo transfer. High-grade embryos were divided between culture and cold storage; low-grade embryos were evaluated only after culture. Over 3 days of culture, both high- and low-grade embryos tended to either maintain or improve upon their initial morphological score (P < 0.05) and increased in diameter (P < 0.001). Embryos subjected to cooling tended to have reduced morphological scores by 48 h of storage and decreased in diameter by 72 h (P < 0.05). No significant influence of storage method (cooling vs. culture), duration (24-72 h), or embryo grade (high vs low) was observed on pregnancy establishment at Day-60 (22.2%-57.2% pregnancy rates for all treatments). Overall, rates of pregnancy establishment were similar for transferred cultured (n = 45) and cooled (n = 45) embryos (pregnancy rates at Day 18, 48% vs 51.1%; at Day 60, 37.7% vs 37.7%). Rates of embryonic loss also were similar (22.7% vs 26%). In conclusion, whilst similar rates of pregnancy and pregnancy loss were observed following the transfer of both cooled and cultured embryos held in vitro for up to 3 days, amongst the two methods, only embryo culture appears to provide a means of effectively preserving Day- 7 dromedary camel embryos with reduced morphological values in vitro. Considering these embryos appear to show poor tolerance to the cooling procedure and are unlikely candidates for vitrification, embryo culture may provide an effective method for deriving pregnancies from low-morphological grade embryos when immediate transfer is not possible on the day of flushing.

Observations of pressure anisotropy effects within semi-collisional magnetized plasma bubbles.

Magnetized plasma interactions are ubiquitous in astrophysical and laboratory plasmas. Various physical effects have been shown to be important within colliding plasma flows influenced by opposing magnetic fields, however, experimental verification of the mechanisms within the interaction region has remained elusive. Here we discuss a laser-plasma experiment whereby experimental results verify that Biermann battery generated magnetic fields are advected by Nernst flows and anisotropic pressure effects dominate these flows in a reconnection region. These fields are mapped using time-resolved proton probing in multiple directions. Various experimental, modelling and analytical techniques demonstrate the importance of anisotropic pressure in semi-collisional, high-ß plasmas, causing a reduction in the magnitude of the reconnecting fields when compared to resistive processes. Anisotropic pressure dynamics are crucial in collisionless plasmas, but are often neglected in collisional plasmas. We show pressure anisotropy to be essential in maintaining the interaction layer, redistributing magnetic fields even for semi-collisional, high energy density physics (HEDP) regimes.

Preparations for a European R&D roadmap for an inertial fusion demo reactor.

A European consortium of 15 laboratories across nine nations have worked together under the EUROFusion Enabling Research grants for the past decade with three principle objectives. These are: (a) investigating obstacles to ignition on megaJoule-class laser facilities; (b) investigating novel alternative approaches to ignition, including basic studies for fast ignition (both electron and ion-driven), auxiliary heating, shock ignition, etc.; and (c) developing technologies that will be required in the future for a fusion reactor. A brief overview of these activities, presented here, along with new calculations relates the concept of auxiliary heating of inertial fusion targets, and provides possible future directions of research and development for the updated European Roadmap that is due at the end of 2020. This article is part of a discussion meeting issue 'Prospects for high gain inertial fusion energy (part 2)'.

Whole-beam self-focusing in fusion-relevant plasma.

Fast ignition inertial confinement fusion requires the production of a low-density channel in plasma with density scale-lengths of several hundred microns. The channel assists in the propagation of an ultra-intense laser pulse used to generate fast electrons which form a hot spot on the side of pre-compressed fusion fuel. We present a systematic characterization of an expanding laser-produced plasma using optical interferometry, benchmarked against three-dimensional hydrodynamic simulations. Magnetic fields associated with channel formation are probed using proton radiography, and compared to magnetic field structures generated in full-scale particle-in-cell simulations. We present observations of long-lived, straight channels produced by the Habara-Kodama-Tanaka whole-beam self-focusing mechanism, overcoming a critical barrier on the path to realizing fast ignition. This article is part of a discussion meeting issue 'Prospects for high gain inertial fusion energy (part 2)'.

Successful vitrification of dromedary camel embryos using a novel embryo vitrification kit.

Embryos (n = 87) collected 8 days after mating and 7 days after ovulation were vitrified using a camel-specific vitrification kit. Vitrification solutions (VS) containing 20% foetal calf serum, with or without 2% bovine serum albumin (BSA) were used to cryopreserve embryos, in three steps VS1 (5 min), VS2 (5 min) and VS3 (30-35 s) at room temperature (RT) before being loaded into open pulled straws and immediately frozen in liquid nitrogen. Embryos were subsequently thawed in warming solutions (WS) in three steps: WS1 at 37 °C (1 min), WS2 at RT (5 min) then into holding media at RT (5-60 min) prior to transfer, in pairs, into recipient camels 6 days after ovulation. There were 42 of 43 embryos viable after vitrification in media without BSA and these were subsequently transferred into 21 recipient females which resulted in ten pregnancies 60 days after transfer (48% pregnancy rate). There were 38 of 44 viable embryos vitrified in media containing BSA that were transferred in pairs into 19 recipient females which resulted in five pregnancies 60 days after transfer (26% pregnancy rate; P > 0.05). Of the total 15 foetuses that developed to 60 days of gestation after vitrification, 11 resulted from embryos of 200-499 µm diameter and four from embryos of 500-700 µm diameter (P > 0.05). There were encouraging results with use of this novel vitrification kit for the commercial application of cryopreservation of camel embryos with a diameter of 300-550 µm.

Preservation of the spermatozoa of the dromedary camel (Camelus dromedarius) by chilling and freezing: The effects of cooling time, extender composition and catalase supplementation.

This study sought to determine the characteristics of dromedary camel sperm following 24 h chilling and cryopreservation, testing two different buffers and cryoprotectants and the presence of catalase (500 IU/mL). Ejaculates were liquefied in Tris-Citric acid-Fructose buffer, and centrifuged through a colloid. For Experiment 1 (n = 5) sperm were cooled 24 h in Green Buffer or INRA-96® containing 0 or 3% glycerol or ethylene glycol. Experiment 2 (n = 5) used the same six treatments to evaluate sperm cryopreserved after 24 h cooling. A test of fertility was run (n = 12 recipients) with split ejaculates of fresh semen cooled 24 h in Green Buffer with and without glycerol. Experiment 3 (n = 7) cryopreserved sperm cooled 2 and 24 h in Green Buffer without cryoprotectant and with and without catalase. Sperm parameters measured before and after treatments included motility, viability and acrosome integrity. Experiment 1 showed no reduction in all sperm parameters after 24 h and no differences between buffers or presence or not of either cryoprotectant. Experiment 2 showed Green Buffer to be better than INRA for supporting sperm frozen after 24 h cooling while, for both buffers, there were few differences in sperm parameters if cryoprotectant was present or absent. Pregnancies were confirmed in 4/6 animals (67%) while no recipients receiving sperm chilled with glycerol were pregnant. In Experiment 3, catalase-supplemented sperm had maintained better motility 2 h post thaw; there were no differences between 2 or 24 h cooled sperm parameters for presence or absence of catalase. There was neither advantage nor disadvantage to coooling sperm 24 h prior to cryopreservation. We concluded that dromedary sperm can be chilled (24 h) and then either inseminated or cryopreserved. While glycerol presence in Green Buffer during chilling did not interfere with cryosurvival it may be toxic to the fertility of fresh chilled sperm. Catalase supplementation during cooling helps maintain sperm motility post thaw.

Collapse dynamics of spherical cavities in a solid under shock loading.

Extraordinary states of highly localised pressure and temperature can be generated upon the collapse of impulsively driven cavities. Direct observation of this phenomenon in solids has proved challenging, but recent advances in high-speed synchrotron radiography now permit the study of highly transient, subsurface events in real time. We present a study on the shock-induced collapse of spherical cavities in a solid polymethyl methacrylate medium, driven to shock states between 0.49 and 16.60 GPa. Utilising multi-MHz phase contrast radiography, extended sequences of the collapse process have been captured, revealing new details of interface motion, material failure and jet instability formation. Results reveal a rich array of collapse characteristics dominated by strength effects at low shock pressures and leading to a hydrodynamic response at the highest loading conditions.

Successful pregnancies from vitrified embryos in the dromedary camel: Avoidance of a possible toxic effect of sucrose on embryos.

Successful embryo cryopreservation facilitates the wider application of assisted reproduction technologies and also provides a useful method for gene banking of valuable genetics. Unfortunately attempts to establish an effective cryopreservation protocol for camelid embryos have been unsuccessful. In the current study, a modified vitrification protocol with three steps was investigated, whereby embryos were exposed to solutions containing increasing amounts of glycerol and ethylene glycol for fixed time periods. Embryos were then loaded into an Open Pull Straw (OPS) and plunged directly into liquid nitrogen for storage. Three experiments were designed to investigate the effect of 1) artificial shrinkage (AS) of embryos, 2) the addition of sucrose to the vitrification solutions, and 3) the replacement of sucrose by galactose in the warming solution, on the outcome of vitrification. The results showed that neither AS of hatched embryos prior to vitrification, nor the addition of sucrose into vitrification solutions improves the outcome of vitrification, while replacement of sucrose with galactose in warming solution increases the survival and developmental rates of vitrified embryos in culture. Transfer of vitrified embryos that were warmed in galactose resulted in a pregnancy rate of 42.8% per embryo or 46.1% per recipient. Collectively, these results suggest a possible species-specific toxic effect of sucrose on camel embryos, and that avoiding its use either in vitrification or warming solution is critical for establishing an effective protocol. This study may also be applicable to the vitrification of embryos of other camelid species including alpaca and llamas.

Absolute calibration of optical streak cameras on picosecond time scales using supercontinuum generation.

We report a new method using high-stability, laser-driven supercontinuum generation in a liquid cell to calibrate the absolute photon response of fast optical streak cameras as a function of wavelength when operating at fastest sweep speeds. A stable, pulsed white light source based around the use of self-phase modulation in a salt solution was developed to provide the required brightness on picosecond time scales, enabling streak camera calibration in fully dynamic operation. The measured spectral brightness allowed for absolute photon response calibration over a broad spectral range (425-650 nm). Calibrations performed with two Axis Photonique streak cameras using the Photonis P820PSU streak tube demonstrated responses that qualitatively follow the photocathode response. Peak sensitivities were one photon/count above background. The absolute dynamic sensitivity is less than the static by up to an order of magnitude. We attribute this to the dynamic response of the phosphor being lower.

Counterpropagating Radiative Shock Experiments on the Orion Laser.

We present new experiments to study the formation of radiative shocks and the interaction between two counterpropagating radiative shocks. The experiments are performed at the Orion laser facility, which is used to drive shocks in xenon inside large aspect ratio gas cells. The collision between the two shocks and their respective radiative precursors, combined with the formation of inherently three-dimensional shocks, provides a novel platform particularly suited for the benchmarking of numerical codes. The dynamics of the shocks before and after the collision are investigated using point-projection x-ray backlighting while, simultaneously, the electron density in the radiative precursor was measured via optical laser interferometry. Modeling of the experiments using the 2D radiation hydrodynamic codes nym and petra shows very good agreement with the experimental results.

Single layer centrifugation of fresh dromedary camel semen improves sperm quality and in vitro fertilization capacity compared with simple sperm washing.

Single layer centrifugation (SLC) through a colloid is a tool for selecting viable mammalian spermatozoa but has not been used previously for fresh dromedary camel sperm. Semen from six camels (2 ejaculates/male) was diluted 1:5 (v:v) or 1:10 (v:v) in a Tris-citrate-fructose buffer for mechanical liquefaction by gentle pipetting. Following liquefaction, semen was processed either by SLC or by centrifugation without a colloid (control). Total and progressive motilities, CASA kinematics, vitality and acrosome integrity (eosin-nigrosin) and plasma membrane integrity (Hypo-osmotic swelling test; HOST), and fertilizing ability in a heterologous assay (zona-free goat oocytes) were evaluated. Both total (p = .003) and progressive motilities (p = .003) were higher in SLC-processed than in control semen samples, irrespective of dilution. Positive HOST values increased when using colloid in 1:5 (p = .001) and 1:10 dilution (p = .010). Colloid-selected sperm had higher penetration rates than controls (p < .001 and p = .02 for 1:5 and 1:10 dilutions, respectively). However, only the SLC sperm at 1:5 dilution showed higher percentages of pronuclear formation (p = .02) than controls. Dilution effect was only significant for total motility before in vitro fertilization, with higher values for the 1:5 dilution (p = .033). The recovery rates of motile sperm between dilutions were similar (26.1% vs 35.4%; p = .226). In conclusion, SLC is a promising tool for selecting functional dromedary camel sperm and warrants more research.

Current status and future direction of cryopreservation of camelid embryos.

Over the past 3 decades, and similar to the horse industry, fresh embryo transfer has been widely practiced on large commercial scales in different camelid species, especially the dromedary camel and alpaca. However, the inability to cryopreserve embryos significantly reduces its broader application, and as such limits the capacity to utilize elite genetic resources internationally. In addition, cryopreservation of the semen of camelids is also difficult, suggesting an extreme sensitivity of the germplasm to cooling and freezing. As a result, genetic resources of camelids must continue to be maintained as living collections of animals. Due to concerns over disease outbreaks such as that of the highly pathogenic Middle East Respiratory Syndrome in the Middle East and Asia, there is an urgent need to establish an effective gene banking system for camelid species, especially the camel. The current review compares and summarizes recent progress in the field of camelid embryo cryopreservation, identifying four possible reasons for the slow development of an effective protocol and describing eight future directions to improve the current protocols. At the same time, the results of a recent dromedary camel embryo transfer study which produced a high morphologic integrity and survival rate of Open Pulled Straw-vitrified embryos are also discussed.

Two-color spatial and temporal temperature measurements using a streaked soft x-ray imager.

A dual-channel streaked soft x-ray imager has been designed and used on high energy-density physics experiments at the National Ignition Facility. This streaked imager creates two images of the same x-ray source using two slit apertures and a single shallow angle reflection from a nickel mirror. Thin filters are used to create narrow band pass images at 510 eV and 360 eV. When measuring a Planckian spectrum, the brightness ratio of the two images can be translated into a color-temperature, provided that the spectral sensitivity of the two images is well known. To reduce uncertainty and remove spectral features in the streak camera photocathode from this photon energy range, a thin 100 nm CsI on 50 nm Al streak camera photocathode was implemented. Provided that the spectral shape is well-known, then uncertainties on the spectral sensitivity limits the accuracy of the temperature measurement to approximately 4.5% at 100 eV.

Laboratory analogue of a supersonic accretion column in a binary star system.

Astrophysical flows exhibit rich behaviour resulting from the interplay of different forms of energy-gravitational, thermal, magnetic and radiative. For magnetic cataclysmic variable stars, material from a late, main sequence star is pulled onto a highly magnetized (B>10 MG) white dwarf. The magnetic field is sufficiently large to direct the flow as an accretion column onto the poles of the white dwarf, a star subclass known as AM Herculis. A stationary radiative shock is expected to form 100-1,000 km above the surface of the white dwarf, far too small to be resolved with current telescopes. Here we report the results of a laboratory experiment showing the evolution of a reverse shock when both ionization and radiative losses are important. We find that the stand-off position of the shock agrees with radiation hydrodynamic simulations and is consistent, when scaled to AM Herculis star systems, with theoretical predictions.

A new streaked soft x-ray imager for the National Ignition Facility.

A new streaked soft x-ray imager has been designed for use on high energy-density (HED) physics experiments at the National Ignition Facility based at the Lawrence Livermore National Laboratory. This streaked imager uses a slit aperture, single shallow angle reflection from a nickel mirror, and soft x-ray filtering to, when coupled to one of the NIF's x-ray streak cameras, record a 4× magnification, one-dimensional image of an x-ray source with a spatial resolution of less than 90 µm. The energy band pass produced depends upon the filter material used; for the first qualification shots, vanadium and silver-on-titanium filters were used to gate on photon energy ranges of approximately 300-510 eV and 200-400 eV, respectively. A two-channel version of the snout is available for x-ray sources up to 1 mm and a single-channel is available for larger sources up to 3 mm. Both the one and two-channel variants have been qualified on quartz wire and HED physics target shots.

Optimization of a vitrification protocol for hatched blastocysts from the dromedary camel (Camelus dromedarius).

The objective of this study was to modify and optimize a vitrification protocol (open pulled straw) that was originally designed for human oocytes and embryos, to make it suitable for the cryopreservation of camel hatched blastocysts. The original open pulled straw protocol was a complex process with 15-minute exposure of oocytes/embryos in 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (Me2SO) for equilibration, and cooling in 16% EG + 16% Me2SO + 1 M sucrose. Recognizing a need to better control the cryoprotectant (CPA) concentrations, while avoiding toxicity to the embryos, the effects on the survival rate and developmental potential of camel embryos in vitro were investigated using two different methods of loading the CPAs into the embryos (stepwise and semicontinuous increase in concentration), two different loading temperature/time (room temperature ∼24 °C/15 min and body 37 °C/3 min), and the replacement of Me2SO with EG alone or in combination with glycerol (Gly). A total of 145 in vivo-derived embryos were subjected to these processes, and after warming their morphological quality and integrity, and re-expansion was assessed after 0, 2, 24, 48, 72, and 96 hours of culture. Exposure of embryos in a stepwise method was more beneficial to the survival of embryos than was the semicontinuous process, and loading of CPAs at 37 °C with a short exposure time (3 minutes) resulted in an outcome comparable to the original processing at room temperature with a longer exposure time (15 minutes). The replacement of the Me2SO + EG mixture with EG only or a combination of EG + Gly in the vitrification medium significantly improved the outcome of all these evaluation criteria (P < 0.05). The modified protocol of loading EG at 37 °C for 3 minutes has increased the embryo survival of the original protocol from 67% to 91% and the developmental rate from 57% to 83% at 5-day culture. These results were comparable to or better than those reported in human or other species, indicating that this optimized method is well suited to any commercial embryo transfer program in the dromedary camel.

Multiwavelength interferometry system for the Orion laser facility.

We report on the design and testing of a multiwavelength interferometry system for the Orion laser facility based upon the use of self-path matching Wollaston prisms. The use of UV corrected achromatic optics allows for both easy alignment with an eye-safe light source and small (∼ millimeter) offsets to the focal lengths between different operational wavelengths. Interferograms are demonstrated at wavelengths corresponding to first, second, and fourth harmonics of a 1054 nm Nd:glass probe beam. Example data confirms the broadband achromatic capability of the imaging system with operation from the UV (263 nm) to visible (527 nm) and demonstrates that features as small as 5 µm can be resolved for object sizes of 15 by 10 mm. Results are also shown for an off-harmonic wavelength that will underpin a future capability. The primary optics package is accommodated inside the footprint of a ten-inch manipulator to allow the system to be deployed from a multitude of viewing angles inside the 4 m diameter Orion target chamber.

Monochromatic radiography of high energy density physics experiments on the MAGPIE generator.

A monochromatic X-ray backlighter based on Bragg reflection from a spherically bent quartz crystal has been developed for the MAGPIE pulsed power generator at Imperial College (1.4 MA, 240 ns) [I. H. Mitchell et al., Rev. Sci. Instrum. 67, 1533 (2005)]. This instrument has been used to diagnose high energy density physics experiments with 1.865 keV radiation (Silicon He-α) from a laser plasma source driven by a ∼7 J, 1 ns pulse from the Cerberus laser. The design of the diagnostic, its characterisation and performance, and initial results in which the instrument was used to radiograph a shock physics experiment on MAGPIE are discussed.

Diagnosing collisions of magnetized, high energy density plasma flows using a combination of collective Thomson scattering, Faraday rotation, and interferometry (invited).

A suite of laser based diagnostics is used to study interactions of magnetised, supersonic, radiatively cooled plasma flows produced using the Magpie pulse power generator (1.4 MA, 240 ns rise time). Collective optical Thomson scattering measures the time-resolved local flow velocity and temperature across 7-14 spatial positions. The scattering spectrum is recorded from multiple directions, allowing more accurate reconstruction of the flow velocity vectors. The areal electron density is measured using 2D interferometry; optimisation and analysis are discussed. The Faraday rotation diagnostic, operating at 1053 nm, measures the magnetic field distribution in the plasma. Measurements obtained simultaneously by these diagnostics are used to constrain analysis, increasing the accuracy of interpretation.

Distribution of MLH1 foci in horse male synaptonemal complex.

Advances in molecular cytogenetics have provided the opportunity to study events during prophase I of meiosis. Immunofluorescent localization of different meiotic protein components were used to characterize the early stages of the first meiotic division in horse spermatocytes. The frequency and distribution of recombination events during prophase I were investigated using the mutL homolog 1 (MLH1) protein that is known to be associated with these events. The frequency and distribution of MLH1 foci were investigated in pachytene nuclei of 6 fertile stallions, and the average relative synaptonemal complex length was found to be highly correlated with the average number of MLH1 foci. The frequency and distribution of MLH1 foci were found to closely correspond to the frequency and distribution of chiasmata on metaphase I chromosomes, and genetic length, calculated from MLH1 foci data, for the whole genome was 2,505.5 cM.