Excess Solution Research Articles

Analysis of Nutritional Status, Body Composition, and Creatinine Levels in Hemodialysis Patients at Ibnu Sina Yw-Umi Hospital Makassar

Hemodialysis is an essential therapy for managing chronic kidney failure, helping to maintain homeostasis by removing excess fluids, solutes, and toxins from the body. The global increase in the prevalence of chronic kidney disease (CKD) has led to a rising demand for hemodialysis. This study aims to analyze the relationship between nutritional status, body composition, and creatinine levels in hemodialysis patients at Ibnu Sina YW-UMI Makassar Hospital. Using an observational cross-sectional design, the study involved 16 patients whose body mass index (BMI), muscle mass, fat mass, total body fluid, and visceral fat were measured before and after dialysis. The results revealed significant changes in nutritional status, body composition, and creatinine levels pre- and post-hemodialysis, analyzed through paired T-tests at a 0.05 confidence level. Most patients were male, aged over 45, with normal nutritional status but abnormal creatinine levels. Body composition analysis showed significant differences in muscle mass, fat mass, total body fluid, and visceral fat before and after hemodialysis, with patients with higher muscle mass tending to have elevated creatinine levels. This study emphasizes the importance of monitoring body composition and creatinine levels to support the health of hemodialysis patients and highlights the need for personalized nutritional and therapeutic interventions in CKD care. Further research is needed to explore the long-term impacts of hemodialysis on nutritional status and kidney function.

A qualitative approach to describe the viscosity of flowable concrete made with manufactured sand containing different microfines

The concrete made with different manufactured sands can exhibit complex rheological properties. The prediction of viscosity is crucial in concrete placement, but remains challenging given the varied content and characteristics of microfines in the sands. This paper studies the influence of tuff (TF) and limestone (LS) microfines with disparate particle size distributions on the pseudo-Newtonian viscosity of concrete prepared with crushed sand. The concrete had a fixed water-to-binder mass ratio of 0.32 and polycarboxylate ether superplasticizer (PCE) dosage of 0.14 % by mass of biner. Test results showed that the microfines in manufactured sand can reduce the aggregate-to-excess cement paste ratio, leading to lower contact interactions between the aggregate grains. But the ratio of total powder particles and entrained air to excess interstitial solutions increased for the fluid paste matrix of the concrete, which caused greater flow resistance related to the powder contacts and air bubble deformations. A qualitative approach correlating the volume ratio of aggregate grains to excess cement pastes and that of total powder particles and entrained air to excess interstitial solutions was proposed to describe the pseudo-Newtonian viscosity of concrete, regardless of the different microfine additions in manufactured sand.

Reverse Solute Diffusion Enhances Sludge Dewatering in Dead-End Forward Osmosis.

Wastewater treatment plants produce high quantities of excess sludge. However, traditional sludge dewatering technology has high energy consumption and occupies a large area. Dead-end forward osmosis (DEFO) is an efficient and energy-saving deep dewatering technology for sludge. In this study, the reverse osmosis of salt ions in the draw solution was used to change the sludge cake structure and further reduce its moisture content in cake by releasing the bound water in cell. Three salts, NaCl, KCl, and CaCl2, were added to the excess sludge feed solution to explore the roles of the reverse osmosis of draw solutes in DEFO. When the added quantities of NaCl and CaCl2 were 15 and 10 mM, respectively, the moisture content of the sludge after dewatering decreased from 98.1% to 79.7% and 67.3%, respectively. However, KCl did not improve the sludge dewatering performance because of the "high K and low Na" phenomenon in biological cells. The water flux increased significantly for the binary draw solute involving NaCl and CaCl2 compared to the single draw solute. The extracellular polymer substances in the sludge changed the structure of the filter cake to improve the formation of water channels and decrease osmosis resistance, resulting in an increase in sludge dewatering efficiency. These findings provide support for improving the sludge dewatering performance of DEFO.

Protective Effects of Small Molecular Inhibitors on Steel Corrosion: The Generation of a Multi-Electric Layer on Passivation Films

The durability of reinforced concrete structures is significantly influenced by the effectiveness of small molecular inhibitors in preventing the corrosion of steel reinforcements. In a concrete environment, the passive film on steel bars serves as a critical protective component. In this study, a molecular dynamics (MD) simulation is used to study the inhibition mechanism of chloride ions by common corrosion inhibitors (2-Amino-2-thiazoline) in concrete in an excess chloride solution. The results reveal that inhibitors adsorb onto the steel surface primarily through van der Waals forces, with more than 90% of the adsorption occurring vertically. Despite this strong adsorption, inhibitors alone do not form a protective film. In the presence of chloride ions, which frequently penetrate concrete, the coverage rate of inhibitors on the steel surface decreases from 74% to 64%. Nevertheless, inhibitor molecules still provide substantial protection in chloride-rich concrete environments. Further analysis indicates that inhibitor molecules inhibit chloride ions in two ways. Corrosion inhibitor molecules actively desorb from the steel surface to capture chloride ions and prevent them from approaching. Additionally, inhibitors form a multi-electron layer on the steel surface to enhance passive film protection and hinder chloride ion diffusion through Coulombic interactions.

Offline constrained reinforcement learning for batch-to-batch optimization of cobalt oxalate synthesis process

The cobalt oxalate synthesis, a batch process, plays a crucial role in the refinement of cobalt metal. The mean particle size of cobalt oxalate is a critical indicator that reflects product quality. However, excessive ammonium oxalate solution flow can heighten waste disposal costs in the production process. To address these issues, we propose a novel offline reinforcement learning (RL) algorithm that guarantees compliance with constraints in the cobalt oxalate synthesis process, utilizing exclusively static datasets. This method employs cost critic networks to assess costs, transforming the constrained optimization problem into an unconstrained one by introducing Lagrangian multipliers. We use exponential moving average (EMA) to optimize the update of proportional integral derivative (PID) control multipliers, reduce overshoot and oscillation in the control process, and thus improve the overall stability of the system. Furthermore, to optimize algorithm performance, a deep residual network (DResNet) is integrated into the policy network. Experimental results indicate that the algorithm’s optimization policy performs significantly better under constraints.

Experimental Study on Macro and Meso Characteristics of Steel-Slag-Based Cemented Backfill Due to Microbial Mineralization Action.

Steel slag is an industrial solid waste, which can provide a new calcium source for microbial mineralization as it contains abundant calcium elements. This study treated cemented backfill material with microorganisms and steel slag to enhance its performance. The influence of microbial treatment on the strength, microstructure, and pore characteristics of the backfill was assessed using a strength test, nuclear magnetic resonance, scanning electron microscopy, and X-ray diffraction. The results indicate that (1) the microbial mineralization and the hydration reaction take place at the same time; (2) when the proportion of bacterial solution exceeded 50%, microorganisms excessively consumed Ca2+, which hindered the following hydration reaction; (3) the additional amount of bacterial solution added into the steel-slag-based cemented backfill material should be less than 50%, which increases the strength by up to 22.10%; (4) the excessive bacterial solution sharply reduces the strength of the backfill even by 21.41%; and (5) the addition of bacterial solution affects the pore characteristics. A 50% bacterial solution can make backfill reach its lowest porosity. The strength has an inversely proportional relationship with porosity, diameter, and roundness (σ = ax + b, a < 0).

Ag nanofilm enhanced S-type Ag@AgCl/tubular g-C3N4/Ti photoanode visible light response photocatalytic fuel cell

In this study, based on Ag@AgCl/tubular g-C3N4 (Ag@AgCl/TCN) composite, Ag nanoparticles were deposited on its surface to form Ag nanofilm enhanced composite through photoreduction of excessive silver nitrate solution to obtain Ag nanofilm Ag@AgCl/TCN photocatalyst. Finally, Ag nanofilm Ag@AgCl/TCN/Ti photoanode was successfully prepared by silica-sol drop-coating method, and assembled with Cu2O/Cu cathode to construct a photocatalytic fuel cell (PFC). Ag nanofilm can not only enhance the light absorption and utilization of Ag@AgCl/TCN/Ti, but also extend its light response wavelength to visible light, and be used as an electron transfer medium to reduce internal resistance. The photocurrent density and the photoconversion efficiency of the Ag nanofilm/Ag@AgCl/TCN/Ti are 8.01 mA·cm−2 and 1.24%, respectively, which are 2.49 and 2.54 times those of the Ag@AgCl/TCN/Ti photoanode, respectively. The first-order kinetic constant and the maximum power density of the Ag nanofilm-coated Ag@AgCl/TCN/Ti-Cu2O/Cu PFC system are 0.064 min−1 and 46.37 μW·cm−2, respectively, which are 1.39 and 1.94 times those of the Ag@AgCl/TCN-Cu2O/Cu PFC system, respectively. In addition, it degraded more than 95% of rhodamine B, 72.68% of tetracycline and 54.25% of bisphenol A within 60 min. The excellent performance of Ag nanofilm/Ag@AgCl/TCN/Ti-Cu2O/Cu is due to the strengthening of Ag nanofilm and the formation of S-type heterojunctions between AgCl and TCN. Free radicals (•Cl, h+, •OH, •O2-) and non-radicals (1O2) are involved in the degradation reaction of RhB degradation. It is worth noting that 6% Ag nanofilm/Ag@AgCl/TCN/Ti-Cu2O/Cu has a positive effect on the production of •O2-, and its output is approximately 1.89 times that of the Ag@AgCl/TCN/Ti-Cu2O/Cu. A series of material characterization experiments have been completed and possible mechanisms of operation have been proposed. This study provides a new scheme for the design and preparation of high-efficiency photoanode of PFC.

Hydrophobic barrier-free laminated paper-based analytical device (LPAD) using a diameter-based measurement for determination of iodide in pharmaceutical products

This work presents, for the first time, an exploitation of an argentometric Mohr's method for iodide detection using diameter-based measurement on laminated paper-based analytical device (LPAD). Our LPAD is simplified fabricated with no required hydrophobic barrier for patterning a detection flow channel. In the absence of a flow channel, the liquid is imbibed radially into the filter paper outward from the small center inlet hole. This imbibition leads to the measurement of diameter after the formation of the precipitation product. For LPAD, a rectangular piece of Whatman filter paper is cut and positioned between a top laminating sheet, which has a hole punched for inlet, and a bottom laminating sheet. The central inlet hole enables access to the liquid on the paper. For iodide analysis, silver nitrate is dispensed first, followed by the iodide standard/sample. This process leads to the formation of pale-yellow silver iodide solids on the paper. The chromate indicator solution is subsequently loaded to the paper. The color of the chromate solution imparts a tint to the circular band of silver iodide solids, resulting in an intense yellow color. When the silver iodide precipitates are stained, the chromate solution typically serves as an indicator, chemically reacting to an excess silver nitrate solution moistened on paper. This reaction generates an outer reddish-brown ring of silver chromate. The distinct color contrast between the intense yellow (tinted silver iodide) and reddish-brown (silver chromate) allows for clear observation of the measurement boundary, facilitating diameter-based measurements of the circular band of silver iodide. We demonstrate the applicability in analysis of pharmaceutical KI tablets. The quantitative results obtained from diameter-based measurement LPAD show good agreement with those obtained from the potentiometric method. Our hydrophobic barrier-free LPAD is rapid, cost-effective, low liquid consumption and applicable for onsite analysis.

Nonionic Microgels Adapt to Ionic Guest Molecules: Superchaotropic Nanoions.

Microgels are commonly applied as solute carriers, where the size, density, and functionality of the microgels depend on solute binding. As representatives for ionic solutes with high affinity for the microgel, we study here the effect of superchaotropic Keggin polyoxometalates (POMs) PW12O403- (PW) and SiW12O404- (SiW) on the aqueous swelling and internal structure of nonionic poly(N-isopropylacrylamide) (pNiPAM) microgels by light scattering techniques and small-angle X-ray scattering. Due to their weak hydration, these POMs bind spontaneously to the microgels at millimolar concentrations. The microgels thus become charged and swell at low POM concentration, surprisingly without strongly increasing the volume phase transition temperature, and deswell at higher POM concentration. The swelling arises because of the osmotic pressure of dissociated counterions of the POMs, while the deswelling is due to POMs acting as physical cross-links in the microgels under screened electrostatics in NaCl or excess POM solution. This swelling/deswelling transition is sharper for PW than for SiW related to the lower charge density, weaker hydration, and stronger binding of PW. The POMs elicit qualitatively and quantitatively different swelling effects from ionic surfactants and classical salts. Moreover, the network softness and topology govern the swelling response upon POM binding. The softer the microgel, the stronger is the swelling response, while, inside the microgel, regions of high polymer density swell/contract more upon electric charging/cross-linking than regions with low polymer density. POM binding thus enables fine-tuning of microgel properties and highlights the role of network topology in microgel swelling. Because POMs decompose at an alkaline pH, these POM/microgel systems also exhibit pH-responsive swelling in addition to the typical temperature responsiveness of pNiPAM microgels.

Microstructure evolution and formation mechanism of interfaces in parallel gap resistance welding of stranded Ag-plated Cu conductor to Ag interconnector

In space flexible solar arrays, stranded Ag-plated Cu conductors and Ag interconnectors are preferred materials for energy transmission. Parallel gap resistance welding is a solderless micro-joining process that favors the reliability of joints between stranded Ag-plated Cu conductors and Ag interconnectors. It was found the interfacial microstructure presented diversity and complexity while mechanical and electrical properties showed unusual nonlinear variations with welding voltage. This study aims to clarify the interfacial microstructure evolution and its effects on the joint properties while elucidating the microstructure formation mechanisms. A shift in the bonding mechanism at interfaces from solid-state diffusion to brazing was found as welding voltage reached a critical value (1.5 V). It eliminated micro gaps and built nano-scale interlocking structures, resulting in substantial improvement in mechanical properties. Increasing the welding voltage from 1.2 V to 1.4 V improved electrical conductivity due to the enlarged bonding area at interfaces. However, high welding voltage (1.6 V) led to degradation in the electrical conductivity of joints due to excessive Ag-Cu solid solution formed at interfaces. The key to fabricating high-strength and high-conductivity joints lies in achieving appropriate interfacial melting while reducing alloying by controlling peak temperature and shortening the duration above the Ag-Cu eutectic point.

Non-equilibrium molecular dynamics of steady-state fluid transport through a 2D membrane driven by a concentration gradient.

We use a novel non-equilibrium algorithm to simulate steady-state fluid transport through a two-dimensional (2D) membrane due to a concentration gradient by molecular dynamics (MD) for the first time. We confirm that, as required by the Onsager reciprocal relations in the linear-response regime, the solution flux obtained using this algorithm agrees with the excess solute flux obtained from an established non-equilibrium MD algorithm for pressure-driven flow. In addition, we show that the concentration-gradient-driven solution flux in this regime is quantified far more efficiently by explicitly applying a transmembrane concentration difference using our algorithm than by applying Onsager reciprocity to pressure-driven flow. The simulated fluid fluxes are captured with reasonable quantitative accuracy by our previously derived continuum theory of concentration-gradient-driven fluid transport through a 2D membrane [D. J. Rankin, L. Bocquet, and D. M. Huang, J. Chem. Phys. 151, 044705 (2019)] for a wide range of solution and membrane parameters, even though the simulated pore sizes are only several times the size of the fluid particles. The simulations deviate from the theory for strong solute-membrane interactions relative to thermal energy, for which the theoretical approximations breakdown. Our findings will be beneficial for a molecular-level understanding of fluid transport driven by concentration gradients through membranes made from 2D materials, which have diverse applications in energy harvesting, molecular separations, and biosensing.

Point excess solute: A new metric for quantifying solute segregation in atom probe tomography datasets including application to naturally aged solute clusters in Al-Mg-Si-(Cu) alloys

Accurate, repeatable and quantitative analysis of nanoscale solute clustering in atom probe tomography (APT) datasets is a complex challenge which is made more difficult by the positional uncertainty and lack of absolute resolution inherent to the technique. In this work a new method, the point excess solute, is introduced for quantifying solute segregation in datasets with limited spatial resolution. This new method is based on measuring the matrix concentration using a dataset sampling method. We show the new method can accurately reproduce the values expected from synthetic datasets a priori and when the dataset spatial resolution and or phase contrast is too low for accurate quantification this is observable. The method is then applied to naturally aged solute clusters in the Al-Mg-Si-Cu system. Datasets were collected with a range of natural ageing times from 8 min to 76 weeks. The formation of the solute clusters is shown to be unaffected by the Cu content of the alloy.

Micro-structured polymer fixed targets for serial crystallography at synchrotrons and XFELs.

Fixed targets are a popular form of sample-delivery system used in serial crystallography at synchrotron and X-ray free-electron laser sources. They offer a wide range of sample-preparation options and are generally easy to use. The supports are typically made from silicon, quartz or polymer. Of these, currently, only silicon offers the ability to perform an aperture-aligned data collection where crystals are loaded into cavities in precise locations and sequentially rastered through, in step with the X-ray pulses. The polymer-based fixed targets have lacked the precision fabrication to enable this data-collection strategy and have been limited to directed-raster scans with crystals randomly distributed across the polymer surface. Here, the fabrication and first results from a new polymer-based fixed target, the micro-structured polymer fixed targets (MISP chips), are presented. MISP chips, like those made from silicon, have a precise array of cavities and fiducial markers. They consist of a structured polymer membrane and a stabilization frame. Crystals can be loaded into the cavities and the excess crystallization solution removed through apertures at their base. The fiducial markers allow for a rapid calculation of the aperture locations. The chips have a low X-ray background and, since they are optically transparent, also allow for an a priori analysis of crystal locations. This location mapping could, ultimately, optimize hit rates towards 100%. A black version of the MISP chip was produced to reduce light contamination for optical-pump/X-ray probe experiments. A study of the loading properties of the chips reveals that these types of fixed targets are best optimized for crystals of the order of 25 µm, but quality data can be collected from crystals as small as 5 µm. With the development of these chips, it has been proved that polymer-based fixed targets can be made with the precision required for aperture-alignment-based data-collection strategies. Further work can now be directed towards more cost-effective mass fabrication to make their use more sustainable for serial crystallography facilities and users.

A recently developed minimum volume, absorbent, vitrification device, the Kitasato Vitrification System gives excellent outcomes for in vitro produced bovine blastocysts

Cryopreservation of embryos is a crucial component of current assisted reproductive technologies (ART). While the ART outcomes for many species have been greatly improved by the introduction of minimum volume vitrification devices, these devices can be difficult to handle and load. To reduce this problem, we recently developed a vitrification carrier which has a highly absorbent surface so that it simply and rapidly removes excess free vitrification solution from the specimen before the cooling step. This Kitasato Vitrification System (KVS) gives excellent results for human and mouse embryo vitrification. This study aimed to determine whether the KVS would also be effective for bovine blastocyst vitrification by comparing outcomes for the control device that was the KVS without excess vitrification solution absorber. The effect of varying the length of time spent in the first equilibration solution (0–10 min) was also evaluated. Vitrification with the KVS resulted in significantly higher survival and hatching rates than with the control device loaded with the same volume of vitrification solution (survival: 98.6% vs 87.6%, hatching at 72 h post warming: 87.3% vs 66.7%, respectively). The best outcomes were obtained with a 10 min equilibration step prior to exposure to the vitrification solution for 30 s. We also evaluated the effect of embryo quality on blastocyst viability when using the KVS. Survival rates of high- and low-quality embryos were comparable but low quality embryos had significantly lower hatching rates. Overall, the results indicate that the KVS vitrification device is effective for bovine blastocyst vitrification.

Achieving ultra-high stiffness by solidifying and precipitating micro-compounds in HCP/BCC dual matrix of a new Mg-Li alloy

One of the major obstacles restricting the wide applications of Mg alloys is their low Young's modulus at ∼45 GPa, which is uncompetitive to Al alloy over 60 GPa. To resolve this issue, we proposed a new strategy of designing ultra-light Mg-Li alloys by employing the microscale compounds in both HCP and BCC grains to achieve high Young’s modulus. HCP or BCC alone cannot achieve a high modulus due to the segregation of solutes at the grain boundaries and the formation of brittle intermetallics in the interdendritic region. We designed chemistry that is so perfect to arrest the excess solutes inside the BCC matrix during solidification and engulf pre-solidified rare earth particles by HCP primary grains which have such a lean chemical composition that has almost no segregation. During solution heat treatment, the dissolution of solutes pushes the BCC boundary towards HCP grains by Ostwald ripening (BCC structures are interconnected but the HCP structures are isolated). During low-temperature aging heat treatment, nucleation of HCP structure on the rare earth particles inside the BCC grain is found, degrading the strength. Therefore, a novel Mg-Li-Al-Zn-Y-Mn-Gd (LAZWMV) alloy with a very high modulus was designed which reaches Young's modulus as high as 61.4 GPa. Micro-compounds (AlLi and Al-X) with average equivalent diameters of 3.76 µm and 6.55 µm were quantified in 3D using X-ray computed tomography and nanoindentation tests for establishing the microstructure-modulus correlation. The influence of Li addition on Mg-Mg was calculated by DFT calculations and the mechanism of modulus improvements has been proposed.

Kinetics of the Saponification of Mixed Fats Consisting of Olein and Stearin

This research presents the kinetics of the saponification reaction using mixed fats of olein and stearin [in the ratio (3:1)] with NaOH solution. In this reaction, excess solution of NaOH was used to ensure the reaction being irreversible. Three parameters were varied to show their effects on the reaction rate .They are: percentage excess of NaOH solution (10 % - 100 %) , temperature (100-150)oC , and stirring speed (400-1100) rpm. It was noticed that increasing the percentage excess of NaOH solution enhances the rate of reaction while increasing temperature decreases the reaction rate since it is exothermic reaction. Increasing stirring speed also improves the reaction rate because it is mass transfer controlled .Calculations of the activation energy and the frequency factor was also performed.&#x0D; A new mathematical model for calculation of the reaction – rate constant was derived. It is shown that a good approximation was obtained between the experimental and calculated values of the reaction – rate constant k .

Preparation and application of a chiral Am7CD-modified COF composite membrane by interfacial polymerization

Membrane separation technology has infinite potential to produce optically pure drugs. However, there are still challenges regarding chiral membranes, such as the single separation object, poor mechanical properties, and constrained relationship between permeability and enantioselectivity. To improve the membrane properties, heptakis (6-amino-6-deoxy)-β-CD-modified carboxyl-functionalized COF (TpBD-Am7CD) was introduced as a chiral functional material for the preparation of a TpBD-Am7CD stainless steel I-Net cellulose acetate membrane (COF/SSCAM) via interfacial polymerization. Using tryptophan as the template molecule, the membrane preparation and filtration conditions were optimized to achieve maximum enantioselectivity. The optimized COF/SSCAM achieved a better enantiomeric excess percentage (e.e.% = 100.00%) and solute permeation flux (JS = 37 × 10−6 mol m−2 h−1) for racemic tryptophan than the support membrane, which was attributed to the enantioselectivity and hydrophilicity of TpBD-Am7CD. Moreover, COF/SSCAM achieved enantiomeric separation of racemates of propranolol, warfarin, atenolol, and metoprolol, and the e.e.% values were 39.51%, 5.60%, 16.04%, and 3.09%, respectively. In addition, the chiral recognition mechanism was investigated by molecular docking simulation.

A Novel Cell Injection Method with Minimum Invasion.

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient amount of suspension solution to allow the cells to enter the tissue. The volume of the suspension solution affects the tissue, and this can cause major invasive injury as a result of the cell injection. This paper reports on a novel cell injection method, called slow injection, that aims to avoid this injury. However, pushing out the cells from the needle tip requires a sufficiently high injection speed according to Newton's law of shear force. To solve the above contradiction, a non-Newtonian fluid, such as gelatin solution, was used as the cell suspension solution in this work. Gelatins solution have temperature sensitivity, as their form changes from gel to sol at approximately 20 °C. Therefore, to maintain the cell suspension solution in the gel form, the syringe was kept cooled in this protocol; however, once the solution was injected into the body, the body temperature converted it to a sol. The interstitial tissue fluid flow can absorb excess solution. In this work, the slow injection technique allowed cardiomyocyte balls to enter the host myocardium and engraft without surrounding fibrosis. This study employed a slow injection method to inject purified and ball-formed neonatal rat cardiomyocytes into a remote area of myocardial infarction in the adult rat heart. At 2 months following the injection, the hearts of the transplanted groups showed significantly improved contractile function. Furthermore, histological analyses of the slow-injected hearts revealed seamless connections between the host and graft cardiomyocytes via intercalated disks containing gap junction connections. This method could contribute to next-generation cell therapies, particularly in cardiac regenerative medicine.

Unilateral Biportal Endoscopy for Lumbar Spinal Stenosis and Lumbar Disc Herniation.

Unilateral Biportal Endoscopy for Lumbar Spinal Stenosis and Lumbar Disc Herniation.

Paradigm shift in frog sperm cryopreservation: reduced role for non-penetrating cryoprotectants.

Sperm cryopreservation has been recognised as a tool for preventing loss of genetic diversity in amphibians; however, the combined effect of penetrative and non-penetrative cryoprotectants in cryodiluents is poorly understood. We demonstrate a clear benefit of using low concentrations of non-penetrative cryoprotectants when cryopreserving sperm of Australian tree frogs. Sperm cryopreservation protocols have been developed for an increasing number of amphibian species since the recognition of a global amphibian decline. Yet, the development of these protocols has neglected to elucidate the combined effect of the penetrative(PCP) and non-penetrative cryoprotectant (NPCP) on the recovery of live, motile sperm. The two-factor hypothesis of cryoinjury recognises a trade-off between cooling cells slowly enough to allow osmotic dehydration and therefore avoid intracellular ice formation, but fast enough to minimise effects from increasing extracellular osmolality as the frozen fraction of the media increases during freezing. We tested the effect of two concentrations of a PCP (10 and 15% v/v dimethyl sulfoxide (Me2SO)) and two concentrations of an NPCP (1 and 10% w/v sucrose) in various combinations on the sperm of six pelodryadid frogs. In all species, 15% v/v Me2SO with 1% w/v sucrose provided superior post-thaw recovery with high proportions of forward progressive motility, live cells and intact acrosomes (typically >50% for each). Theoretically, it has been suggested that increased NPCP concentration should improve cell survival by increasing the rate and extent of cell dehydration. We suggest, however, that the elevated osmolality in the unfrozen water fraction when 10% sucrose is used may be causing damage to cells via excessive cell shrinkage and solute effects as proposed in the two-factor hypothesis of cryoinjury. We showed this response in sperm across a range of frog species, providing compelling evidence for this hypothesis. We suggest protocol development using the PCP/NPCP ratios demonstrated in our study will be broadly applicable to many amphibian species.