Vitrification of germinal-vesicle stage equine oocytes: Effect of cryoprotectant exposure time on in-vitro embryo production

Previous studies have found low rates of blastocyst development (0–11%) after vitrification of germinal vesicle (GV)-stage equine oocytes. In this study, we systematically evaluated a short (non-equilibrating) system for GV-stage oocyte vitrification. In Exp. 1, we assessed oocyte volume in cumulus-oocyte complexes (COCs) exposed to components of a short protocol, using 2% each of ethylene glycol and propylene glycol in the first solution (VS1); 17.5% of each plus 0.3 M trehalose in the second solution (VS2); and fetal bovine serum as the base medium. Based on the time to oocyte minimum volume, we selected a 40-sec exposure to VS1. In Exp. 2, we evaluated exposure times to VS2 and, based on rates of subsequent maturation in vitro, we selected 65 s. In Exp. 3, we used the optimized vitrification system (40-VS1; 65-VS2) and evaluated three warming procedures. Blastocyst development after ICSI was equivalent (15%) for COCs warmed in either standard (trehalose stepwise dilution) or isotonic (base medium) solutions, but was reduced (0%) for COCs warmed in a highly hypertonic (1.5 M trehalose) solution. Exposure to the vitrification and warming solutions, without actual vitrification, was associated with reduced blastocyst development (0–5%; Exp. 4). We conclude that this optimized short protocol supports moderate blastocyst production after vitrification of GV-stage equine COCs. Oocytes can be warmed in isotonic medium, which simplifies the procedure. The systems used still showed a high level of toxicity and further work is needed on both vitrification and warming methods to increase the efficiency of this technique.     

Effects of vegetation canopy and climate on seedling establishment in Mediterranean shrubland

Abstract. Question: Does the influence of plant canopy on seedling establishment interact with climate conditions, and particularly, do intensified drought conditions, enhance a positive effect of the vegetation canopy on seedlings in Mediterranean‐type ecosystems. Location: Mediterranean shrubland near Barcelona, Spain at 210 m a.s.l. Methods: Over the course of four years we recorded seedling emergence and survival in open areas and below vegetation under control, drier and warmer experimental climatic conditions. Results: Seedling emergence is more sensitive to climate conditions than later stages of growth. When considering the whole set of species, the total number of established seedlings at the end of the experiment was lower in the drought and warming stands than in control ones, and vegetation canopy increased the number of these seedlings in the drought stands. Drought reduced seedling emergence but not warming, while the interaction between climate treatments and vegetation canopy was not significant. Seedling survival was lower in the warming treatment than in the control. Under drought conditions, vegetation canopy increased seedling emergence of the dominant Globularia alypum. In control stands, vegetation canopy reduced their survival. Vegetation canopy increased the survival of the dominant Erica multiflora in warming stands, and it reduced the survival of G alypum in drought stands. No significant effects of drought and warming were observed in the seed rain of these two species. Conclusions: The balance of the facilitation‐competition interactions between vegetation canopy and seedling establishment in Mediterranean‐type ecosystems determined by water availability, and drought conditions enhance the positive effect of vegetation canopy. This interaction is species‐specific and shows important between‐year variability.     

滋补肾阴方与温补肾阳方对卵巢切除所致骨质疏松大鼠治疗作用的对比研究

目的　对比观察滋补肾阴方与温补肾阳方对卵巢切除所致骨质疏松大鼠的治疗作用 ,以探讨它们之间的疗效是否存在差异。方法　将雌性Wistar大鼠 4 9只随机分为正常对照组、假手术组、卵巢切除组、滋阴组 (灌服生药含量为 1 0 5g ml的滋补肾阴方 ,按每 10 0g体重 0 7ml剂量 )、补阳组 (灌服生药含量为 0 78g ml的温补肾阳方 ,按每 10 0g体重 0 7ml剂量 )。于造模 3个月后开始给药 ,1次 日 ,连续 6d ,休息一天后 ,再连续灌服 6d。给药 2个月后 ,将大鼠处死 ,取胫骨行不脱钙骨切片 ,甲苯胺蓝染色 ,对其进行骨组织形态计量。结果　切除卵巢 5个月后 ,大鼠胫骨TBV %显著降低 ,TRS %以及TFS %、AFS %、MAR、BFR、OSW和mAR皆显著增高 ,给大鼠灌服滋阴补肾方和温补肾阳方 2个月后 ,均能使上述指标发生逆转 ,但程度有所不同 ,温补肾阳方的效果要明显优于滋补肾阴方。结论　滋补肾阴方与温补肾阳方对卵巢切除所致的骨质疏松均有明显的治疗作用 ,但温补肾阳方的疗效要优于滋阴补肾方。     

Extreme rapid warming yields high functional survivals of vitrified 8-cell mouse embryos even when suspended in a half-strength vitrification solution and cooled at moderate rates to −196 °C

To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to do so is to subject them to procedures that convert cell water into a non-crystalline glass. Current belief is that to achieve this vitrification, cells must be suspended in very high concentrations of glass-inducing solutes (i.e., ?6 molal) and cooled at very high rates (i.e., ?1000 °C/min). We report here that both these beliefs are incorrect with respect to the vitrification of 8-cell mouse embryos. In this study, precompaction 8-cell embryos were vitrified in several dilutions of EAFS10/10 using various cooling rates and warming rates. Survival was based on morphology, osmotic functionality, and on the ability to develop to expanded blastocysts. With a warming rate of 117,500 °C/min, the percentages of embryos vitrified in 1×, 0.75×, and 0.5× EAFS that developed to blastocysts were 93%, 92%, and 83%, respectively. And the percentages of morphological survivors that developed to expanded blastocysts were 100%, 92%, and 97%, respectively. Even when the solute concentration of the EAFS was reduced to 33% of normal, we obtained 40% functional survival of these 8-cell embryos.     

Climate change and Epichloë coenophiala association modify belowground fungal symbioses of tall fescue host

Human alteration of symbiont genetics among aboveground endophytic Epichloë coenophiala strains within tall fescue (Schedonorus arundinaceus) has led to widespread deployment of novel grass-endophyte combinations, yet little is known about their ecological consequences. In this study, clone pairs (endophyte-infected, endophyte-free) of two tall fescue genotypes received factorial combinations of increased temperature (+3 °C) and precipitation (+30% long-term annual mean) for 2 yr. We measured root arbuscular mycorrhizal fungi (AMF), dark septate endophyte (DSE) colonization, and soil AMF extraradical hyphae (ERH) length. We hypothesized that genetically distinct grass-E. coenophiala associations would differentially affect belowground fungi, and that these relationships would be climate-sensitive. Tall fescue genotype, endophyte presence, and climate treatment interactions affected AMF arbuscules, vesicles, and ERH. DSE decreased with E. coenophiala presence but increased with warming. Genetically distinct tall fescue-E. coenophiala associations may have divergent long-term impacts on other host-symbiont interactions, potentially impacting ecosystem function and response to climate change.     

Is it getting hot in here? Adjustment of hydraulic parameters in six boreal and temperate tree species after 5 years of warming

Global temperatures (T) are rising, and for many plant species, their physiological response to this change has not been well characterized. In particular, how hydraulic parameters may change has only been examined experimentally for a few species. To address this, we measured characteristics of the hydraulic architecture of six species growing in ambient T and ambient +3.4 °C T plots in two experimentally warmed forest sites in Minnesota. These sites are at the temperate–boreal ecotone, and we measured three species from each forest type. We hypothesized that relative to boreal species, temperate species near their northern range border would increase xylem conduit diameters when grown under elevated T. We also predicted a continuum of responses among wood types, with conduit diameter increases correlating with increases in the complexity of wood structure. Finally, we predicted that increases in conduit diameter and specific hydraulic conductivity would positively affect photosynthetic rates and growth. Our results generally supported our hypotheses, and conduit diameter increased under elevated T across all species, although this pattern was driven predominantly by three species. Two of these species were temperate angiosperms, but one was a boreal conifer, contrary to predictions. We observed positive relationships between the change in specific hydraulic conductivity and both photosynthetic rate (P = 0.080) and growth (P = 0.012). Our results indicate that species differ in their ability to adjust hydraulically to increases in T. Specifically, species with more complex xylem anatomy, particularly those individuals growing near the cooler edge of their range, appeared to be better able to increase conduit diameters and specific hydraulic conductivity, which permitted increases in photosynthesis and growth. Our data support results that indicate individual's ability to physiologically adjust is related to their location within their species range, and highlight that some wood types may adjust more easily than others.     

Injury due to extravasation of thiopental and propofol: Risks/effects of local cooling/warming in rats

Inadvertent leakage of medications with vesicant properties can cause severe necrosis in tissue, which can have devastating long-term consequences. The aim of this study was to evaluate the extent of extravasation injury induced by thiopental and propofol, and the effects of cooling or warming of local tissue on extravasation injury at macroscopic and histopathologic levels. Rats were administered intradermally thiopental (2.5 mg/100 µL) or propofol (1.0 mg/100 µL). Rats were assigned randomly to three groups: control (no treatment), cooling and warming. Local cooling (18–20 °C) or warming (40–42 °C) was applied for 3 h immediately after agent injection. Lesion sizes (erythema, induration, ulceration, necrosis) were monitored after agent injection. Histopathology was evaluated in skin biopsies taken 24 h after agent injection. Thiopental injection induced severe skin injury with necrosis. Peak lesions developed within 24 h and healed gradually 18–27 days after extravasation. Propofol induced inflammation but no ulceration, and lesions healed within 1–2 days. Local cooling reduced thiopental- and propofol-induced extravasation injuries but warming strongly exacerbated the skin lesions (e.g., degeneration, necrosis) induced by extravasation of thiopental and propofol. Thiopental can be classified as a “vesicant” that causes tissue necrosis and propofol can be classified as an “irritant”. Local cooling protects (at least in part) against skin disorders induced by thiopental and propofol, whereas warming is harmful.     

Effect of chilling on sox2, sox3 and sox19a gene expression in zebrafish (Danio rerio) embryos

Zebrafish embryos have not been cryopreserved due to their structural limitations. Although embryo survival rates have been used as the measured outcome for most of the cryopreservation protocols studied, there are very limited data available at the molecular level. This study focused on the effect of chilling and subsequent warming on gene expression of sox2, sox3 and sox19a which play vital roles in the development of zebrafish embryos. A quantitative RT-PCR approach was used to investigate gene expression following chilling at 0 °C for up to 180 min. The effect on gene expression was also studied during a 180 min warming period after chilling for 30 or 60 min. There were significant decreases in sox2 (up to 4-fold) and sox3 (up to 3-fold) expressions following chilling. Significant increases in gene expressions of sox2 (up to 2-fold), sox3 (up to 33-fold) and sox19a (up to 25-fold) were observed during warming in the embryos that had been chilled for 30 min. Similarly, significant increases were observed in sox2 (up to 3-fold) and sox3 (up to 2-fold) during warming in embryos that had been chilled for 60 min. These increases may be explained by compensation for the suppression observed during chilling and/or to activate repair mechanisms or maintain homeostasis.     

Differences among dogs in response of their spermatozoa to cryopreservation using various cooling and warming rates

Spermatozoa collected from the caudae epididymides of 16 dogs of various breeds were suspended in an isotonic salt solution (DIMI medium) containing 0.6 M glycerol, frozen in liquid nitrogen, and their "survival" was measured after thawing. In the first experimental series, duplicate samples of spermatozoa from each of 11 dogs were cooled at rates of 0.5, 3, 11, 58, or 209 degrees C/min, stored in liquid nitrogen, and the frozen samples warmed at approximately 830 or at 33 degrees C/min. Sperm "survival" was judged by microscopic assessments of motility and of membrane integrity, the latter as assayed with Fertilight, a double fluorescent stain. Motility of frozen spermatozoa that were thawed rapidly, averaged for 11 dogs, was low at low rates, increased to a maximum at 11 degrees C/min, and then decreased significantly at higher rates (P<0.01). This inverted V-shaped curve was also observed with slow thawing, although the apparent optimum cooling rate ranged from 3 to 11 degrees C/min. The integrity of sperm plasma membranes showed a similar dependence on cooling rate, although the percentages of spermatozoa with intact plasma membranes were higher than the percentages of motile spermatozoa. Motility of spermatozoa, as a function of cooling rate, varied considerably from male to male (P<0.01), whereas membrane integrity was much more consistent among the 11 dogs. In the second experimental series with spermatozoa from 5 dogs, motility of spermatozoa frozen at 0.5 degrees C/min and warmed at 3.6, 33, 140, or 830 degrees C/min also exhibited an inverted V-shaped survival curve, in this case as a function of warming rate. In summary, high survival of frozen-thawed canine epididymal spermatozoa depended on both cooling and warming rates, but spermatozoa from each dog exhibited their own sensitivity to cooling and warming rates.     

Soil Solution Chemistry and Element Fluxes in Three European Heathlands and Their Responses to Warming and Drought

Soil water chemistry and element budgets were studied at three northwestern European Calluna vulgaris heathland sites in Denmark (DK), The Netherlands (NL), and Wales (UK). Responses to experimental nighttime warming and early summer drought were followed during a two-year period. Soil solution chemistry measured below the organic soil layer and below the rooting zone and water fluxes estimated with hydrological models were combined to calculate element budgets. Remarkably high N leaching was observed at the NL heath with 18 and 6.4 kg N ha^–1 year^–1 of NO₃–N and NH₄–N leached from the control plots, respectively, indicating that this site is nitrogen saturated. Increased soil temperature of +0.5°C in the heated plots almost doubled the concentrations and losses of NO₃–N and DON at this site. Temperature also increased mobilization of N in the O horizon at the UK and DK heaths in the first year, but, because of high retention of N in the vegetation or mineral soil, there were no significant effects of warming on seepage water NO₃–N and NH₄–N. Retention of P was high at all three sites. In several cases, drought increased concentrations of elements momentarily, but element fluxes decreased because of a lower flux of water. Seepage water DOC and DON was highly significantly correlated at the UK site where losses of N were low, whereas losses of C and N were uncoupled at the NL site where atmospheric N input was greatest. Based on N budgets, calculations of the net change in the C sink or source strength in response to warming suggest no change or an increase in the C sink strength during these early years.