Developing a platform for Fresnel diffractive radiography with 1 µm spatial resolution at the National Ignition Facility.

An x-ray Fresnel diffractive radiography platform was designed for use at the National Ignition Facility. It will enable measurements of micron-scale changes in the density gradients across an interface between isochorically heated warm dense matter materials, the evolution of which is driven primarily through thermal conductivity and mutual diffusion. We use 4.75 keV Ti K-shell x-ray emission to heat a 1000 µm diameter plastic cylinder, with a central 30 µm diameter channel filled with liquid D2, up to 8 eV. This leads to a cylindrical implosion of the liquid D2 column, compressing it to ∼2.3 g/cm3. After pressure equilibration, the location of the D2/plastic interface remains steady for several nanoseconds, which enables us to track density gradient changes across the material interface with high precision. For radiography, we use Cu He-α x rays at 8.3 keV. Using a slit aperture of only 1 µm width increases the spatial coherence of the source, giving rise to significant diffraction features in the radiography signal, in addition to the refraction enhancement, which further increases its sensitivity to density scale length changes at the D2/plastic interface.

Diffraction enhanced imaging utilizing a laser produced x-ray source.

Image formation by Fresnel diffraction utilizes both absorption and phase-contrast to measure electron density profiles. The low spatial and spectral coherence requirements allow the technique to be performed with a laser-produced x-ray source coupled with a narrow slit. This makes it an excellent candidate for probing interfaces between materials at extreme conditions, which can only be generated at large-scale laser or pulsed power facilities. Here, we present the results from a proof-of-principle experiment demonstrating an effective ∼2 µm laser-generated source at the OMEGA Laser Facility. This was achieved using slits of 1 × 30 µm2 and 2 × 40 µm2 geometry, which were milled into 30 µm thick Ta plates. Combining these slits with a vanadium He-like 5.2 keV source created a 1D imaging system capable of micrometer-scale resolution. The principal obstacles to achieving an effective 1 µm source are the slit tilt and taper-where the use of a tapered slit is necessary to increase the alignment tolerance. We demonstrate an effective source size by imaging a 2 ± 0.2 µm radius tungsten wire.

Toward an integrated platform for characterizing laser-driven, isochorically heated plasmas with 1 µm spatial resolution.

Warm dense matter is a region of phase space that is of high interest to multiple scientific communities ranging from astrophysics to inertial confinement fusion. Further understanding of the conditions and properties of this complex state of matter necessitates experimental benchmarking of the current theoretical models. We discuss the development of an x-ray radiography platform designed to measure warm dense matter transport properties at large laser facilities such as the OMEGA Laser Facility. Our platform, Fresnel diffractive radiography, allows for high spatial resolution imaging of isochorically heated targets, resulting in notable diffractive effects at sharp density gradients that are influenced by transport properties such as thermal conductivity. We discuss initial results, highlighting the capabilities of the platform in measuring diffractive features with micrometer-level spatial resolution.

Seminal plasma addition attenuates the dilution effect in bovine sperm.

Dilution of semen to low cell numbers/dose can result in a bull-dependent reduction in the post-thaw viability of cryopreserved bovine spermatozoa. It is possible that essential seminal plasma components are lacking at the greater dilution rates, thereby contributing to the deleterious effects of semen dilution. Ejaculates of 6 Holstein bulls were diluted to 120 x 10(6) sperm/mL in an egg yolk citrate extender (EYC). Split samples were further diluted to 80, 40, 20 and 4 x 10(6) sperm/mL in EYC extender with (+SP) and without (-SP) the addition of frozen/thawed seminal plasma previously obtained from a vasectomized bull. Serial dilutions for the +SP treatments were calculated and performed such that each dilution contained a volume of seminal plasma equal to the original 120 x 10(6) sperm/mL dilution. Samples were then loaded into 0.5-mL French straws yielding final sperm concentrations of 30, 20, 10, 5 and 1 x 10(6)/dose. Straws from each dilution were analyzed using 2 stain combinations: the sperm viability stain, SYBR-14 and propidium iodide (PI); or the mitochondrial-specific, membrane potential-dependent stain JC-1 along with PI. Split-plot analysis of variance indicated that within bulls, there were greater proportions of viable spermatozoa in aliquots containing added seminal plasma than in aliquots without added seminal plasma (P < 0.05). Contrast analyses showed that sperm viability significantly decreased as sperm concentration decreased in the -SP samples. Although the dilution effect was also observed in the +SP samples, the magnitude of the effect was less than in the -SP samples. At most sperm concentrations, the proportions of spermatozoa that stained with JC-1 were correlated (r > 0.84; P < 0.05) with the percentages of SYBR- 14 stained spermatozoa. Furthermore, the proportions of JC-1-stained spermatozoa were greater in the +SP aliquots than in the -SP samples at a concentration of 10 x 10(6) sperm/0.5 mL. These results suggest that the addition of seminal plasma can be beneficial to sperm viability when semen is diluted to low cell numbers/dose.

An evidence-based approach to management of increased intracranial pressure.

Current treatment of many conditions associated with elevated ICP of the brain involves stabilization and oxygenation with maintenance of adequate perfusion of cerebral tissue, while maintaining an acceptable ICP. As an example of a standard protocol that is in concordance with what is already known about a patient with a severe head injury, the first priority is radiographic screening for a surgical lesion. Further treatment, as shown by the previously outlined studies, includes keeping the patient normothermic, normoglycemic, and normocapnic, and placing an indwelling ICP monitor. Acutely elevated ICP is treated with mannitol, and if this fails, patients are routinely sedated, paralyzed, and mildly hyperventilated, while repeat radiology is obtained to rule out a further surgical lesion. Hypothermia, aggressive hyperventilation, and barbiturate coma continue to be used and are reserved for intractable ICP elevation, or as warranted based on a specific patient (Table 2).

Effect of semen dilution on bovine sperm viability as determined by dual-DNA staining and flow cytometry.

Living and dead spermatozoa were examined for the effects of sperm concentration level on sperm viability. Semen was collected from two different bulls on each of four collection dates. A ninth bull was collected on all four collection dates as a control for effects of collection date. The ejaculates from these nine bulls were diluted to 30 x 10(6) spermatozoa/0.5 ml and then serially diluted to 20, 10, 5, or 1 x 10(6) spermatozoa/0.5 ml French straw. One-half of the straws for each dilution series was stored 24 hours at 5 degrees C, while the other half was cryopreserved. Spermatozoa were stained with SYBR-14 and propidium iodide (PI) to assess viability. Flow cytometry yielded dot plots showing three distinct sperm populations: dead red-stained spermatozoa (PI), viable green-stained spermatozoa (SYBR-14), and moribund spermatozoa that stained both red and green (doubly-stained). Populations were expressed and analyzed in terms of mean percentage of viable spermatozoa and by actual numbers of viable spermatozoa per insemination dose. The mean percentage of living spermatozoa decreased linearly with decreasing sperm concentration; whereas the decrease was parabolic when those same samples were expressed as the mean number of living spermatozoa per insemination dose. The percentage of SYBR-14-stained spermatozoa differed among concentration levels and among bulls (P < 0.01). There were no differences among straws from the same ejaculate. The total volume of ejaculated semen and the concentration of spermatozoa in that ejaculate were both significantly positively correlated with the percentage of SYBR-14-stained spermatozoa in that semen when it was cryopreserved and diluted to < 10 x 10(6) spermatozoa/0.5 ml. In contrast, there were no significant correlations between the initial ejaculate characteristics and the proportion of SYBR-14-stained spermatozoa in the 24-hour-stored samples at any concentration. In conclusion, the percentage of viable spermatozoa in an ejaculate significantly decreased with increasing dilution. Further, in cryopreserved samples, the percentage of living spermatozoa < 10 x 10(6) spermatozoa/0.5 ml depended on the original volume and the sperm concentration of that particular ejaculate.

Comparison of seminal quality in Holstein bulls as yearlings and as mature sires.

Semen quality was compared in 5 Holstein bulls from samples collected as young sires (yearlings) and again as mature bulls after a mean interval of 1,265 d. At both sampling periods, the semen was examined for ejaculate volume, sperm numbers, post-thaw progressive motility and sperm viability. Sperm viability was assessed on cryopreserved samples with fluorescent SYBR-14 to stain living spermatozoa and propidium iodide (PI) to identify dead spermatozoa. The fluorescent populations of stained spermatozoa were quantified by flow cytometry. The percentages of living spermatozoa for the individual bulls, as determined by green fluorescence of SYBR-14, ranged from 44 +/- 3.1 to 54 +/- 0.3 for yearlings, and from 38 +/- 1.5 to 55 +/- 1.0 for mature sires. No differences in sperm viability were found between samples taken from yearling bulls and those of mature bulls. The percentage of spermatozoa stained with SYBR-14 was negatively correlated (r = -0.97; P = 0.0001) with the percentage of dead spermatozoa as indicated by PI staining. Comparisons of identical samples run on 2 different flow cytometers indicated that either flow instrument could be used to assess sperm viability. Although the individual bulls differed (P < 0.05) in ejaculate volume and sperm numbers as yearlings, they did not differ in these parameters as mature bulls. The average number of spermatozoa per ejaculate changed as a result of maturation, increasing from 6.2 +/- 1.0 to 10.7 +/- 1.1 x 10(9). Aging was significantly correlated with ejaculate volume (r = 0.76; P = 0.01) but not with the total number of spermatozoa per ejaculate (r = 0.51; P = 0.13). The maturational changes that occurred in the 5 bulls were minimal with the exception of the increased volume of the ejaculate and the number of spermatozoa per ejaculate.

Fertility of bull semen packaged in .25- and .5-milliliter french straws.

The fertility of bull semen packaged in .25- and .5-mL french straws was compared. One ejaculate from each of five Holstein bulls was split, extended to 10 x 10(6) spermatozoa/inseminate dose in whole homogenized milk, packaged in .25- and .5-mL french straws, frozen in liquid nitrogen (LN) vapor, and stored in LN. Semen was thawed at 37 degrees C for 30 s. Synchronized heifers (n = 1,360) were inseminated (during a 12-mo period) with semen packaged in either a .25- or .5-mL french straw. Blood was collected on the day of insemination and the serum was assayed for progesterone. Heifers with blood progesterone levels of > 1 ng/mL were eliminated from the data. Blood was collected at 30 to 45 d after insemination and the serum was assayed for the presence of bovine pregnancy-specific protein B (bPSPB) by RIA to determine pregnancy. Conception was 63.6 and 62.0% (P = .55) for semen packaged in the .25- and .5-mL french straws, respectively. There was neither a bull x packaging unit interaction (P = .49) nor a day of insemination x packaging unit interaction (P = .87). Conception among bulls ranged from 57.1 to 68.0% (P = .19). No evidence was found that meteorological factors influenced conception. Under the conditions of this experiment, semen packaged in the .25- and .5-mL french straw had similar fertility.

Timing of artificial insemination of dairy cows: fixed time once daily versus morning and afternoon.

Nonreturn rates to professional technician service of 7240 first AI Holstein cows were calculated to evaluate differences between once daily and a.m.-p.m. AI. To determine whether management practices affected nonreturn rates, participating herd owners were surveyed for methods used for detection of estrus. Nonreturn rates for once daily and a.m.-p.m. AI were 64.6 and 65.6% for 60-d, 60.1 and 60.6% for 75-d, and 58.4 and 57.8% for 90-d nonreturn periods. Signs of estrus for AI and interval from detection of estrus to AI were related to nonreturn rates. Nonreturn rate was highest, 63.4%, when cows were in standing estrus. Nonreturn rates were lowest, 36%, when cows were bred after treatment with PGF2 alpha without being detected in estrus or bred strictly on veterinary advice based on palpation. Nonreturn rates were similar for different times of the day when once daily AI was practiced. However, AI in the midmorning may have some advantages. The highest nonreturn rate for a 3-h period was 68.2% for 0800 and 1100 h; the lowest was 54.7% for 1300 to 1600 h. Movement before observation for estrus and an observation period > 15 min improved nonreturn rates for once daily AI. Once daily AI can be used effectively with no difference from the traditional a.m.-p.m. system; results are best when AI is based on standing estrus and performed between 0800 and 1100 h.

Fluorometric evaluation of cryopreserved bovine spermatozoa extended in egg yolk and milk.

A comparison of fluorogenically quantifiable parameters of cryopreserved, bovine spermatozoa that had been processed in homogenized milk and egg yolk citrate-based extenders was made using flow cytometry. Semen from four bulls was processed in egg yolk-citrate or homogenized milk extenders, packaged in straws and frozen at -196 degrees C. Samples were thawed at 37 degrees C, subdivided into three portions and stained after 0, 1.5 and 3 h of incubation at 37 degrees C. Spermatozoa were stained using a combination of carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) and analyzed by dual parameter flow cytometry. The sperm cells were quantified fluorometrically at each time interval for both green and red fluorescence. The proportion of spermatozoa retaining the fluorescent CFDA derivative was larger at each time interval for samples in egg yolk citrate than those in milk. Differences in the retention of spermatozoal viability were detected between identical samples of bovine spermatozoa extended in milk or egg yolk based media.

Effects of extender and insemination dose on postthaw quality and fertility of bovine sperm.

Quality and fertility of sperm extended in egg yolk-Tes-Tris were compared with those of sperm in egg yolk-citrate and homogenized milk extenders. Extended semen was frozen in .5-ml plastic straws at 11, 15, 17, or 22 X 10(6) sperm per insemination dose. Laboratory evaluations at 0, 1, 2, and 4 h after thawing semen utilized four tests of spermatozoal quality. Use of egg-yolk citrate extender resulted in a higher percentage of progressively motile sperm as determined visually at 0 h after thawing than use of egg yolk-Tes-Tris or homogenized milk extenders. Sperm extended in egg yolk-citrate had 18% lower activity of bound amidase at 0 h than sperm extended in egg yolk-Tes-Tris. The 75-d nonreturn rates were affected by insemination dose but not be extender or the interaction of extender and insemination dose. Fertility was lower after insemination of 11 X 10(6) sperm than for pooled data for the three higher insemination doses (64 vs. 68%). Based on all data, postthaw quality of sperm processed in the one-step egg yolk-Tes-Tris extender was similar to that for sperm extended in egg yolk-citrate or homogenized milk.

Efforts to correlate laboratory with field observations on bull sperm fertility.

This work represents efforts towards development of the zona-free hamster ovum sperm penetration assay for predicting relative levels of fertility for semen from individual bulls. Results reported here followed insemination of hamster vitelli with bull sperm, after frozen storage, with observations of sperm acrosomes and parallel inseminations of more than 1000 cows with semen from each bull. The average 75-day non-return rate for the four bulls was 74.0% (range 71.6 to 75.6). Laboratory studies indicated the following: the percentage of sperm with intact acrosomes varied from 55 to 73 between bulls, the percentage of motile sperm varied from 41 to 64, the percentage of sperm with progressive motility ranged from 24 to 40, the number of sperm interacting per (zona-free hamster) ovum ranged from 1.6 to 3.8, the number of sperm attached per ovum ranged from 1.4 to 2.9, the number of sperm within each penetrated ovum ranged from 1.5 to 1.8, the percentage of ova interacting with sperm ranged from 76 to 92, the percentage of ova penetrated ranged from 62 to 85, and the percentage of ova with male pronuclei ranged from 33 to 49. Although predictive ranking in the laboratory of these bulls with less than 4% variation in fertility levels was not possible, the zone-free hamster ovum test could be useful in identifying potentially subfertile bulls before they enter a young sire-sampling program.

Effect on fertility of freezing large numbers of straws of bovine spermatozoa in a mechanical freezer.

Fertility of semen frozen in a mechanical forced-vapor freezer was compared with that frozen in static nitrogen (N) vapor. Semen from 10 Holstein bulls was extended in milk-10% glycerol at 30 x 10(6) progressively motile spermatozoa/ml and packaged in .5-ml French straws. Straws were divided into three equal groups . bull-1 . collection day-1 and frozen in a mechanical freezer at full or half loads (7,250 +/- 250 or 3,500 +/- 250 straws in 5-straw goblets on metal canes) or in static N vapor (330 +/- 30 straws held singly on horizontal racks). For field distribution, straws frozen in static N vapor also were placed in 5-straw goblets on canes after being frozen. Each of the two goblets on a cane held a different treatment. Based on 75-d nonreturn rates for 23,137 first service inseminations, fertility was 1.6 percentage points higher (P less than .05) for semen frozen in static vapor (66.3%) than that frozen in a fully loaded mechanical freezer (64.7%). The difference of 1.3 percentage points in fertility favoring static vapor over freezing a half load in a mechanical freezer (65.0%) approached significance (P = .06); half and full loads did not vary (P greater than .05). In conclusion, the small increase in fertility favoring static over forced-vapor freezing supports use of the static vapor method unless the savings in time and labor of freezing large numbers of straws at one time in a mechanical freezer are considered to offset the small difference in fertility.

Effect of bulk freezing straws of bovine spermatozoa in a programmed freezer on post - thaw survival.

Twelve ejaculates were extended both in skimmilk and Tris-yolk, packaged in .5-ml French straws and frozen vertically in bulk (10,000 straws/freeze) in a programmed mechanical freezer (Linde CRFC-3). Cooling rates and post-thaw spermatozoal survival in the upper and lower thirds of straws at the following six chamber positions were compared: corner and middle within-goblet positions at center, intermediate and outer chamber locations. Cooling rates generally were faster in the upper third of straws than in the lower third and at the corner rather than the middle within-goblet positions. For combined postthaw incubation periods of 0, 3 and 6 hr at 37 C, motility (photographic and visual) and acrosomal retention (fixed and unfixed samples) were both higher (P greater than .01) for sperm in each extender in the upper third of straws than in the lower third. Spermatozoal motility and acrosomal retention generally were higher (P greater than .05) in each extender at the center rather than at the outer chamber location. Among within-goblet positions, post-thaw survival of sperm was higher (P greater than .05) at the corner than at the middle positions for Tris-yolk but not skimmilk. For comparison semen was frozen in static N vapor in straws held singly (258-straw load) on horizontal racks. For combined incubation periods, post-thaw survival of sperm did not differ (P less than .05) between bulk and static vapor- systems. There also was no difference when each of the six chamber positions was compared individually with static vapor. In conclusion, 33% more straws of semen can be frozen in a mechanical freezer than previously reported, with post-thaw spermatozoal survival comparable to that of sperm in straws frozen conventionally in static vapor.