Heterogeneous Associative Plasticity in the Auditory Cortex Induced by Fear Learning – Novel Insight Into the Classical Conditioning Paradigm

We used two-photon calcium imaging with single-cell and cell-type resolution. Fear conditioning induced heterogeneous tuning shifts at single-cell level in the auditory cortex, with shifts both to CS+ frequency and to the control CS- stimulus frequency. We thus extend the view of simple expansion of CS+ tuned regions. Instead of conventional freezing reactions only, we observe selective orienting responses towards the conditioned stimuli. The orienting responses were often followed by escape behavior.

Optimizing the conventional method of sperm freezing in liquid nitrogen vapour for Wallachian sheep conservation program

The aim of the present study was to optimize the conventional method of sperm freezing in liquid nitrogen (LN₂) vapour for successful cryopreservation of Wallachian ram sperm, the genetic resources of the Czech Republic. Sperm in straws were frozen using the conventional freezing method via a static exposure of sperm doses to LN₂ vapour, or by four different modified freezing methods. Under modified freezing, straws were frozen by a discontinuous, time-dependent decremental change in the distance between the straws and the surface of LN₂. The viability of sperm was evaluated by flow cytometry after sperm equilibration, and immediately after thawing. Besides the observed inter-sire and daily variation, the obtained results suggest the methodological weakness of the conventional freezing method via the static exposure of sperm doses to LN₂ vapour. With the use of the optimized freezing procedure, all parameters of thawed sperm were significantly (P < 0.05) improved in comparison with the conventional method: percentage of thawed sperm viability increased up to 48.3%, percentage of sperm with plasma membrane damage after thawing decreased to 6.58%, percentage of sperm with acrosome damage decreased to 24.4%, and percentage of sperm with deteriorated mitochondrial activity decreased to 6.28%. In conclusion, our results suggest that an optimized freezing procedure should be routinely used instead of the conventional method to cryopreserve Wallachian ram sperm.

Frozen strawberry quality enhancement using high hydrostatic pressure and vacuum infusion with pectin methylesterase and calcium chloride solution

Fresh strawberry(Fragaria x ananassa) is a delicious fruit that is an excellent source of micronutrients and an array of beneficial phytochemicals. Emerging technologies like high pressure freezing and thawing have gained interest recently due to their ability to preserve the structure of the food during storage, albeit the possible shortcomings of degradation of texture and sensory quality. In this study, the effects of high hydrostatic pressure freezing and thawing on the physical quality attributes (texture, drip loss and Degree of Methylation(DM)) of strawberry were investigated. Strawberry samples were infused in a vacuum using pectin methyl esterase(PME) and CaCl2solutionat170 hPa pressure for 5 min at room temperature to improve textural integrity; infused with distilled water; and another group of samples further incubated at 40°C for 20 min. Fresh samples and non-pre treated samples were also studied. All the samples were subjected to high pressure freezing and thawing processes at 200 MPa and the effect of the processing conditions on texture were evaluated. The high pressure processed samples were compared with conventionally frozen and thawed samples.Pectin methyl esterase,CaCl2infusion and incubation gave a relative hardness of 1.5 pre treatment compared to water only infused at 0.5.TheCaCl2and PME-infused samples had a higher relative hardness after one day of storage at -18°C compared to non-pretreated and water-infused samples at 0.3 compared to 0.1,respectively. Pretreated, water-infused, and PME withCaCl2-infused samples showed no significant difference in hardness when measured immediately after conventional thawing and high pressure induced thawing HPIT but are the former are a third less hard compared to the latter upon storage after 3 hours and three months. Galacturonic acid per mg of Alcohol insoluble residues AIR sample for the fresh strawberrywas0.002±2.699E-05 and 0.002±1.070E-05molesfor the one-day vs.three months storage,respectively,for samples conventionally frozen and thawed in duplicate experiments.Therefore,a combination of high-pressure shift freezing (HPSF) with PME and CaCl2infusion improved the texture of the strawberry compared to those that were not pretreated,and gained weight by 15%. The PME and CaCl2-infused strawberry showed less degradation than the non-pretreated and water-infused during both long-and short-time frozen storage. Conventional freezing processes caused more degradation compared to HPSF. Degradation of 3 months conventional freezing was comparable to 1-day storage. For frozen storage, enzyme pretreatment combined with high pressure freezing can be used to enhance the quality of strawberry.

Vitrification of Donkey Sperm: Is It Better Using Permeable Cryoprotectants?

Vitrification by direct exposure of sperm to liquid nitrogen is increasing in popularity as an alternative to conventional freezing. In this study, the effect of permeable cryoprotectant agents for donkey sperm vitrification was compared to an extender containing non-permeable cryoprotectants. First, three different concentrations of sucrose (0.1, 0.2, and 0.3 molar, M) and bovine serum albumin, BSA (1, 5, and 10%) were compared. Secondly, the concentration of non-permeable agents producing the most desirable results was compared to an extender containing glycerol as permeable agent. Vitrification was performed by dropping 30 μL of sperm suspension directly into LN2 and warming at 42 °C. Sperm motility (total, TM; and progressive, PM) and plasma membrane integrity, PMI (mean ± SEM) were statistically compared between treatments. Sucrose 0.1 M showed a significantly higher percentage of total sperm motility (21.67 ± 9.22%) than sucrose 0.2 M (14.16 ± 4.50%) and 0.3 M (8.58 ± 6.22%); and no differences were found in comparison to the control (19.71 ± 10.16%). Vitrification with sucrose 0.1 M or BSA 5% obtained similar results for TM (21.67 ± 9.22% vs. 19.93 ± 9.93%), PM (13.42 ± 6.85% vs. 12.54 ± 6.37%) and PMI (40.90 ± 13.51% vs. 37.09 ± 14.28); but both showed higher percentages than glycerol (TM = 9.71 ± 4.19%; PM = 5.47 ± 3.17%; PMI = 28.48 ± 15.55%). In conclusion, donkey sperm vitrification in spheres using non-permeable cryoprotectants exhibited better sperm motility and viability parameters after warming than sperm vitrification using extenders containing permeable cryoprotectants.

Research and Application of the Local Differential Freezing Technology in Deep Alluvium

Aiming at the complicated engineering conditions of the auxiliary shaft repair in the Banji coal mine, it was proposed to seal the water around the shaft lining by differential control freezing technology using double rows of holes. The outer row of holes is completely frozen, and the inner row of holes is local differential frozen according to the degree of destruction of the shaft lining. The local differential freezing pipe was successfully developed according to engineering requirements. Numeral simulations were used to predict the development of the freezing temperature field; the results showed that the inward expansion range of the frozen wall formed by the inner row freezing holes was effectively limited and the temperature drop rate of the shaft lining was significantly reduced after the local differential freezing technique was adopted. The on-site monitoring data showed that the temperature of the limited freezing layer was about 5°C higher than that of the conventional freezing layer. During the drainage work and the construction of the new shaft lining, the thickness and average temperature of the frozen wall remained stable, indicating that the implementation of the local differential freezing technology achieved the expected results. Further analysis showed that when the temperature of the limited freezing part of freezing pipes in the inner row was controlled within the range of −15 to −10°C, not only could the frozen wall reach the design thickness and strength but the frost heaving pressure on the existing shaft lining could be effectively eliminated.

Effect of warming temperatures on donkey sperm vitrification in 0.5 mL straws in comparison to conventional freezing

Aim of study: There is little information about vitrification of sperm in large volumes (up to 0.5 mL). This study aimed to develop the vitrification technique in 0.5 mL straws in donkey sperm, evaluating the effect of three warming temperatures.Area of study: Cordoba, Spain.Material and methods: Ejaculates from five donkeys were divided in four groups: one control subjected to conventional slow freezing (C) and three vitrified in 0.5 mL straws and warmed using different protocols (W1: 37ºC/30s, W2: 43ºC/20s and W3: 70ºC/8s+37ºC/52s). Sperm motility, kinematic parameters, plasma membrane and acrosome integrity were evaluated. Conventional freezing resulted in significantly higher values for total (42.7±19.6%), and progressive motility (30.3±16.7%), plasma membrane (49.1±10.4%) and acrosome integrity (39.6±14.5%) respect to vitrification method.Main results: Values after warming ranged between 0.2-2.8% for total motility; 0.2-2.1% for progressive motility; 5.5-20.0% for plasma membrane integrity and 14.5-29.8% for acrosome integrity in all warming protocols after sperm vitrification. However, no differences were found between W3 and C for kinematic parameters; and W3 resulted in significantly higher values for membrane integrity (20.0±11.0%) in comparison to W1 (5.5±3.6%) and W2 (9.3±8.4%).Research highlights: High warming rates seem to be better for donkey sperm vitrification in large volumes; but this methodology is still not an alternative to conventional sperm freezing.