Optimal Osmolality Research Articles

A test for plasticity in sperm motility activation in response to osmotic environment in an anuran amphibian.

Evolutionary theory predicts that selection will favor phenotypic plasticity in sperm traits that maximize fertilization success in dynamic fertilization environments. In species with external fertilization, osmolality of the fertilization medium is known to play a critical role in activating sperm motility, but evidence for osmotic‐induced sperm plasticity is limited to euryhaline fish and marine invertebrates. Whether this capacity extends to freshwater taxa remains unknown. Here, we provide the first test for plasticity in sperm‐motility activation in response to osmotic environment in an anuran amphibian. Male common eastern froglets (Crinia signifera) were acclimated to either low (0 mOsmol kg−1) or high (50 mOsmol kg−1) environmental osmolality, and using a split‐sample experimental design, sperm were activated across a range of osmolality treatments (0, 25, 50, 75, 100, and 200 ± 2 mOsmol kg−1). Unexpectedly, there was no detectable shift in the optimal osmolality for sperm‐motility activation after approximately 13 weeks of acclimation (a period reflecting the duration of the winter breeding season). However, in both the low and high acclimation treatments, the optimal osmolality for sperm‐motility activation mirrored the osmolality at the natural breeding site, indicating a phenotypic match to the local environment. Previously it has been shown that C. signifera display among‐population covariation between environmental osmolality and sperm performance. Coupled with this finding, the results of the present study suggest that inter‐population differences reflect genetic divergence and local adaptation. We discuss the need for experimental tests of osmotic‐induced sperm plasticity in more freshwater taxa to better understand the environmental and evolutionary contexts favoring adaptive plasticity in sperm‐motility activation.

Comparative methodological studies of L-asparaginase encapsulation into erythrocytes

Background. L-asparaginase is an enzyme, widely used in the therapy of acute lymphoblastic leukemia in children and adults, but its use is limited due to a wide range of side effects and anaphylactic reactions. L-asparaginase loaded into erythrocytes can solve these problems. This enzyme is protected from the immune system and plasma proteases due to erythrocyte membrane, but continues to work inside the cell because its membrane is permeable to L-asparagine. Thus, the half-life of the drug increases and anaphylactic reactions reduce. The encapsulation of L-asparaginase into erythrocytes can be performed by various osmotic methods. Each of them is characterized by the amount of encapsulated enzyme, the cell yield, as well as by the quality indices of the survived erythrocytes. An important parameter of each method is the possibility to provide sterility of this dosage form for the clinical use.The aim of the study was the comparing of three osmotic methods of L-asparaginase encapsulation into erythrocytes (hypo-osmotic lysis, dialysis and flow dialysis) to select the most promising method for clinical use.Materials and methods. A suspension of erythrocytes of healthy donors (hematocrit 60–70%) was mixed with L-asparaginase from E. сoli. The procedures of hypotonic reversible lysis, dialysis in dialysis bags, or flow dialysis using pediatric dialyzers were performed. The physiological osmolality was restored in suspensions after the procedure by the addition of a hypertonic solution, and they were incubated for 30 min at 37 °C. Then the cells were washed in isotonic phosphate-buffered saline with pH 7.4. Activity of L-asparaginase, volume, hematocrit, hematological indices and osmotic cell fragility of erythrocytes were measured in the suspensions of erythrocytes before and after the enzyme encapsulation procedure.Results. An optimal osmolality of the hypotonic buffer for each method was selected and was equal to 90–110 mOsm/kg. The yields of encapsulation were 4.2 ± 2.0, 6.0 ± 2.3 and 16.2 ± 2.2 % for hypotonic lysis, dialysis and flow dialysis, respectively. The hematological indices of the obtained erythrocyte-carriers differed from the corresponding parameters of the initial erythrocytes, but did not differ significantly for different methods.Conclusion. Comparative investigation of mentioned above parameters allowed choosing the method of flow dialysis as the most promising for clinical use.

Parallel substrate supply and pH stabilization for optimal screening of E. coli with the membrane-based fed-batch shake flask

BackgroundScreening in the fed-batch operation mode is essential for biological cultivations facing challenges as oxygen limitation, osmotic inhibition, catabolite repression, substrate inhibition or overflow metabolism. As a screening tool on shake flask level, the membrane-based fed-batch shake flask was developed. While a controlled supply of a substrate was realized with the in-built membrane tip, the possibilities for replenishing nutrients and stabilizing pH values was not yet exploited. High buffer concentrations were initially used, shifting the medium osmolality out of the biological optimum. As the growth rate is predefined by the glucose release kinetics from the reservoir, the resulting medium acidification can be compensated with a controlled continuous supply of an alkaline compound. The focus of this research is to establish a simultaneous multi-component release of glucose and an alkaline compound from the reservoir to enable cultivations within the optimal physiological range of Escherichia coli.ResultsIn combination with the Respiratory Activity MOnitoring System, the membrane-based fed-batch shake flask enabled the detection of an ammonium limitation. The multi-component release of ammonium carbonate along with glucose from the reservoir resulted not only in the replenishment of the nitrogen source but also in the stabilization of the pH value in the culture medium. A biomass concentration up to 25 g/L was achieved, which is one of the highest values obtained so far to the best of the author’s knowledge with the utilization of a shake flask and a defined synthetic medium. Going a step further, the pH stabilization allowed the decrease of the required buffer amount to one-fourth establishing an optimal osmolality range for cultivation. As optimal physiological conditions were implemented with the multi-component release fed-batch cultivation, the supply of 0.2 g glucose in a 10 mL initial culture medium volume with 50 mM MOPS buffer resulted in a twofold higher biomass concentration than in a comparable batch cultivation.ConclusionsThe newly introduced multi-component release with the membrane-based fed-batch shake flask serves a threefold purpose of replenishing depleted substrates in the culture medium, stabilizing the pH throughout the entire cultivation time and minimizing the necessary amount of buffer to maintain an optimal osmolality range. In comparison to a batch cultivation, these settings enable to achieve higher biomass and product concentrations.

Sperm motility activation in the critically endangered booroolong frog: the effect of medium osmolality and phosphodiesterase inhibitors.

Effective activation of sperm motility is fundamental to successful artificial fertilisation; however, studies investigating optimal procedures in amphibians are lacking. This study found the optimal osmolality of activation media for sperm motility activation and evaluated the effect of phosphodiesterase (PDE) inhibitors on sperm activation and longevity in the critically endangered booroolong frog, Litoria booroolongensis. To assess the effect of medium osmolality (10, 25, 50, 75, 100 and 200mOsmolkg-1) and PDE inhibitors (control, 2.5mM caffeine, 5mM caffeine, 2.5mM pentoxifylline, 5mM pentoxifylline, 2.5mM theophylline and 5mM theophylline) on initial activation, percentage sperm motility and sperm velocity were quantified using computer-assisted sperm analysis. To assess the effect of PDE inhibitors (control, 2.5mM caffeine and 2.5mM theophylline) on sperm longevity, percentage motility and velocity were assessed hourly until 10h after activation. High (>60%) percentage motility was achieved in a broad range of activation-medium osmolalities (10-75mOsmolkg-1). PDE inhibitors did not have an effect on initial sperm motility or velocity, but caffeine and theophylline improved sperm longevity, significantly increasing motility and velocity at 8, 9 and 10h after activation. Data also show that sperm longevity in L. booroolongensis is extreme, with spermatozoa remaining motile more than twice as long as those of any other anuran amphibian.

Interpretation of osmotic gradient ektacytometry (osmoscan) data: A comparative study for methodological standards

Osmotic gradient ektacytometry (measuring elongation index in the function of osmolality at a constant shear stress) is a sensitive method to analyze red blood cell (RBC) deformability and investigating the optimal osmolality range for the cells in normal or pathophysiological cellular and micro-environmental conditions. However, the methodological conditions are different, since the results are influenced by the applied shear stress (SS). In this study we investigated rat, dog, pig and human blood samples at SS of 1, 2, 3, 5, 10, 20 and 30 Pa. To describe the range being related to the cell deformability, we introduced new calculated parameters obtained from the raw data of the elongation index (EI)-osmolality (O) curves. Our results showed that: (1) Osmoscan data tested at 20 or 30 Pa do not differ significantly from each other; (2) Under SS of 20 Pa the EImax, the O (EImax), the EI min and the area under curve nearly linearly decrease in the function of SS with different slope in rat, dog, pig and human blood; (3) Measurements under 3 Pa SS become unstable; (4) The differences between minimal and maximal EI and the belonging osmolality values, and their ratios, as new calculated parameters (ΔEI, ΔO, ΔEI/ΔO, EImax/EImin and O (EImax)/Omin) can be suitable for further analysis of the osmoscan curves together with other hemorheological parameters describing RBC deformability; and (5) Decreased erythrocyte deformability (by rigidifying with glutaraldehyde) can be reflected well with the following, calculated osmoscan parameters: ΔO, rO, rEI/rO and ΔEI/ΔO.

The effectiveness of oral rehydration solution at various concentrations as a storage media for avulsed teeth.

Following avulsion, the periodontal ligament (PDL) cells are at risk of necrosis. To achieve a favorable survival prognosis, the PDL cells must be kept viability. Therefore, immediate replantation is considered as the treatment of choice and in case it is not possible, storing the tooth in an appropriate storage media should be considered. Oral Rehydration Solution (ORS) is a glucose-electrolyte solution which can keep the optimal osmolality as well as pH and can even provide nutrients which are necessary for cellular growth. The present study aimed to evaluate the effectiveness of different concentrations of ORS in maintaining the viability of the PDL cells at different time points. PDL cells were obtained from healthy extracted human premolars. Then, 8×10³ cells were seeded in each well of 96-well plate. Afterwards, each well was treated with ORS in three different concentrations and DMEM for 1, 3, 6, and 9 hours. Cell viability was determined by using the MTT assay. One way-ANOVA and post hoc (LSD) test were used for comparing the study groups. According to the results, 25% and 50% concentrations of ORS were more effective in preserving the PDL cell viability and could maintain 79.98% and 68.34% of the PDL cells, respectively, at least for the last experimental time point (up to 9 hours). Therefore, our findings indicate that ORS might be a suitable storage medium for avulsed teeth.

Artificial fertilisation in a terrestrial toadlet (Pseudophryne guentheri): effect of medium osmolality, sperm concentration and gamete storage

Anurans exhibit a greater reproductive diversity than any other vertebrate order. However, studies investigating the effects of the external fertilisation environment on fertilisation success are limited to aquatic-breeding species. This study investigated the effects of fertilisation medium osmolality, sperm concentration and short-term oocyte storage on fertilisation success in a terrestrial-breeding anuran, Pseudophryne guentheri. Split-clutch experimental designs were used to determine optimal fertilisation conditions. To determine the effect of short-term sperm storage, sperm viability was assessed using fluorescence microscopy and percentage sperm motility and velocity quantified with a computer-assisted sperm analysis system. Fertilisation success was highest in media ranging in osmolality from 25 mOsm kg⁻¹ to 100 mOsm kg⁻¹, representing a broader range and higher optimal osmolality than previously reported for aquatic breeders. High rates of fertilisation (>75%) were achieved in relatively low sperm concentrations (2.5×10⁴ mL⁻¹). Oocytes stored in isotonic solutions (200 mOsm kg⁻¹) retained fertilisation capacity (32%) after 8h of storage, while sperm suspensions maintained motility (≥26%) for 13 days. Additional studies on terrestrial-breeding anurans will be required to ascertain whether the optimal fertilisation conditions reported reflect adaptations to achieve fertilisation in a terrestrial environment.

Reassessing culture media and critical metabolites that affect adenovirus production

Adenovirus production is currently operated at low cell density because infection at high cell densities still results in reduced cell-specific productivity. To better understand nutrient limitation and inhibitory metabolites causing the reduction of specific yields at high cell densities, adenovirus production in HEK 293 cultures using NSFM 13 and CD 293 media were evaluated. For cultures using NSFM 13 medium, the cell-specific productivity decreased from 3,400 to 150 vp/cell (or 96% reduction) when the cell density at infection was increased from 1 to 3 x 10(6) cells/mL. In comparison, only 50% of reduction in the cell-specific productivity was observed under the same conditions for cultures using CD 293 medium. The effect of medium osmolality was found critical on viral production. Media were adjusted to an optimal osmolality of 290 mOsm/kg to facilitate comparison. Amino acids were not critical limiting factors. Potential limiting nutrients including vitamins, energy metabolites, bases and nucleotides, or inhibitory metabolites (lactate and ammonia) were supplemented to infected cultures to further investigate their effect on the adenovirus production. Accumulation of lactate and ammonia in a culture infected at 3 x 10(6) cells/mL contributed to about 20% reduction of the adenovirus production yield, whereas nutrient limitation appeared primarily responsible for the decline in the viral production when NSFM 13 medium was used. Overall, the results indicate that multiple factors contribute to limiting the specific production yield at cell densities beyond 1 x 10(6) cells/mL and underline the need to further investigate and develop media for better adenoviral vector productions.

A high yield of fetal nucleated red blood cells isolated using optimal osmolality and a double‐density gradient system

To increase the yield of fetal nucleated red blood cells (NRBCs) from maternal blood using a discontinuous Percoll gradient and to determine the effects of osmolality on NRBC yield. Fetal NRBCs were isolated from combined umbilical cord blood and adult female blood, or from maternal blood using single or double Percoll gradients with different osmolalities. Magnetic activated cell sorting was used to enrich isolated NRBCs, and morphological differentiation was performed with Kleihauer-Betke stain. We also isolated fetal NRBCs from 25 10 mL samples of maternal blood and determined fetal sex by fluorescence in situ hybridization (FISH), using X-Y probes. For single-density Percoll columns, the greatest number of NRBCs was isolated using 280 mOsm/kg H(2)O with 1.077 g/mL Percoll and 520 mOsm/kg H(2)O with 1.119 g/mL Percoll. Significantly more fetal NRBCs were isolated with double Percoll density gradients than with double-Histopaque gradients (p = 0.043). FISH analysis on NRBC in 25 cases correctly identified 15 male and 9 female euploid fetuses and one Trisomy 21 fetus. The NRBC enrichment method we present requires less maternal blood and yields more NRBCs compared to previous methods.

Extender osmolality and sugar supplementation exert a complex effect on the cryopreservation of Iberian red deer ( Cervus elaphus hispanicus) epididymal spermatozoa

We have carried out two experiments to study the cryobiology of red deer epididymal spermatozoa and to improve freezing extenders: (1) effect of extender (Tris-citrate–fructose) osmolality (300–600 mOsm/kg), and (2) effect of sugar (0.4 M) supplementation to the extender (no sugar, glucose, fructose, mannose, sucrose, maltose, threalose and raffinose). Sperm quality was assessed pre-freezing, post-thawing, and after 2 h at 37 °C post-thawing: sperm motility index (SMI), acrosome integrity and membrane integrity (HOS test) were assessed subjectively; mitochondrial activity (JC-1) and membrane stability (merocyanine 540) were assessed by flow cytometry. In experiment 2, DNA status was assessed using acridine orange and flow cytometry. To find an optimal osmolality, we fitted the data to a quadratic curve. Four hundred Osmolal per kilogram rendered better results pre-freezing and post-thawing. However, post-thawing viability and most parameters after 2 h incubation fitted a linear model. Osmolalities above 425 mOsm/kg were deleterious ( P < 0.05). In experiment 2, fructose had a positive effect respect to control after 2 h of incubation at 37 °C post-thawing. Di and trisaccharides were deleterious. Trehalose showed impaired DNA status after 2 h incubation. In conclusion, the osmolality of the extenders should be around 400 mOsm/kg, although the change from quadratic to lineal may indicate a complex effect that must be further studied. Monosaccharides may enhance red deer epididymal sperm cryopreservation, especially fructose, whereas di and trisaccharides may not be adequate.

Effectiveness of a tris-based extender (SHOTOR diluent) for the preservation of Bactrian camel ( Camelus bactrianus) semen

The development of a suitable semen extender is required to extend artificial breeding programs and to preserve the genetic potential of Bactrian camel. Experiments were conducted to provide the optimal osmolality and pH of tris-based extender and to compare that with available extenders for short-term preservation of Bactrian camel semen at 4 °C during 24 h. In experiments I and II, the effects of varying osmolalities (270, 300, 330, 360, and 390 mOsm/kg) and pHs (5.5, 6, 6.9, 7.5, 7.9, and 8.9) of tris-based extender on sperm viability were investigated. In experiment III, the efficiency of tris-based extender (SHOTOR diluent) in preserving Bactrian camel semen was compared with lactose (10%), sucrose (10%) and Green buffer. Viability parameters including progressive forward motility (PFM), plasma membrane integrity and the percentage of live spermatozoa were assessed. The data were analyzed using general linear model procedure. In the majority of assessments using tris-based extender, the viability of spermatozoa was superior at the osmolality of 330 mOsm/kg and pH of 6.9. PFM was significantly greater at the time of semen dilution in tris-based (65.5%) and Green buffer (60.5%) compared to that of lactose (31%) and sucrose (28%) extenders ( P < 0.05), and remained elevated throughout the experiment. There was no significant difference in other viability parameters among 4 extenders ( P > 0.05). In conclusion, the utilization of a tris-based extender, having the osmolality of 330 mOsm/kg and pH of 6.9, favors the short-term preservation of the Bactrian camel spermatozoa under chilled condition.

Respiration in the muscle mitochondria of the mudskipper, Boleophthalmus boddaerti

1. 1. Morphologically and biochemically intact muscle mitochondria were isolated from Boleophthalmus boddaerti. 2. 2. The optimal osmolality was observed to be 300 mOsm which is higher than osmolality (250–270 mOsm) normally used for other organisms. 3. 3. Oxidative phosphorylation in the muscle mitochondria of B. boddaerti was stimulated by glutamate, glutamine, α-ketoglutarate, malate and succinate, while citrate, isocitrate and fumarate were not effective substrates. 4. 4. Phosphorylation in the muscle mitochondria was inhibited by 1 mM 2,4-dinitrophenol, 0.2 μM antimycin A and 1 mM KCN. 5. 5. Rotenone inhibited phosphorylation in all substrates tested except succinate. 6. 6. Oxygen consumption in the presence of a substrate and ADP was stimulated by the uncoupling agent FCCP. 7. 7. The presence of the three sites of coupling in the electron transport system of B. boddaerti was confirmed but coupling in Sites 2 and 3 was not as efficient as in Site 1. 8. 8. It can be concluded that the metabolism of the muscle mitochondria of B. boddaerti, apart from some minor differences, is similar to that of other organisms studied.

Optimal osmolality for cold storage of the cardiac explant

Cell swelling is a major problem of cardiac preservation. This study evaluated the effect of storage solution osmolality on long-term preservation of cardiac function. Isolated rat hearts were flushed and stored in solutions of 260 to 350 mOsm/kg water at 0°C for 9 hr. Cardiac performance was assessed using the isolated working heart reperfusion. Function of fresh unstored hearts served as the control. The stored hearts had normal heart rate after reperfusion. Other hemodynamic functions recovered to various levels of the control value and showed biphasic responses to solution osmolality. Hearts stored in hyposmotic (260 and 270 mOsm) and hyperosmotic (310, 330, and 350 mOsm) solutions performed poorly. Those stored in 280 to 290 mOsm solutions showed superior recovery in cardiac function. Among all parameters, coronary flow correlated linearly with aortic flow, cardiac output, systolic pressure, work, oxygen consumption, and coronary vascular resistance. Solutions of 280, 290, and 310 mOsm caused 6 to 9% increases in tissue water (TW) over the control hearts during 9 hr storage, whereas 350 mOsm solution maintained TW at the control level. After reperfusion, all stored hearts gained water (25 to 38%) compared to hearts with no reperfusion. Postreperfusion myocardial ATP content was only 70–79% of control level and did not correlated to function recovery. In conclusion, (1) the optimal osmolality of our preservation fluid is 280 to 290 mOsm and moderate or severe hyperosmolality is detrimental to cardiac function, and (2) preservation of coronary function may be crucial to future improvement of storage solution.

Effect of osmolality on fertilization of mouse and golden hamster eggs in vitro.

Fertilization of mouse and hamster eggs in vitro in media of various calculated osmolalities was investigated. A modified Krebs\x=req- Ringer bicarbonate solution with bovine serum albumin was used for the mouse gametes and a modified Tyrode's solution with heated hamster blood serum was used for the hamster gametes. Penetration of mouse eggs could occur in media, the osmolality of which varied from 256 to 418 mosmolal, but the optimal osmolality was between 308 and 372 mosmolal. Penetration of hamster eggs could occur in media with osmolality varying from 232 to 452 mosmolal but the optimal osmolality was from 292 to 392 mosmolal. It seems that fertilization in vitro could be achieved better in hypertonic than hypotonic solutions and that hamster gametes could withstand the variation of osmolality slightly better than mouse gametes.