Different Concentrations Of Trehalose Research Articles

Trehalose and glycerol have a dose-dependent synergistic effect on the post-thawing quality of ram semen cryopreserved in a soybean lecithin-based extender

The objective of this study was to examine the interaction of different concentrations of trehalose [0 (T0), 50 (T50) or 100 (T100) mM] and glycerol [5% (G5) or 7% (G7)] on post-thawed quality of ram semen, cryopreserved in a soybean lecithin (SL)-based extender. Twenty-eight ejaculates were collected from four rams and diluted with six trehalose/glycerol combinations: T0G5, T50G5, T100G5, T0G7, T50G7, and T100G7. Sperm motility (CASA), membrane integrity (eosin/nigrosin) and functionality (HOST), abnormal forms, capacitation status (CTC), mitochondrial activity (rhodamine 123), apoptotic features (Annexin V/propidium iodide) and lipoperoxidation (malondialdehyde production) were evaluated after thawing. Extender T100G5 yielded the highest results for total and progressive motility, sperm velocity, normal morphology, functional membranes, active mitochondria and membrane integrity, with P<0.05 in general, except for T50G7 (P>0.05). The combinations T0G5, T0G7 and T100G7 yielded the lowest post-thaw quality. We could not detect significant changes in other kinematic parameters, capacitation status or lipoperoxidation. We conclude that, in our SL-based extender, a combination of 100mM trehalose and 5% glycerol was the most adequate combination to achieving post-thawing quality in our soybean lecithin-based extender, and our results support that a synergistic effect among trehalose and glycerol exists. We suggest that other combinations could improve these results.

Inducible trehalase enzyme activity of Morchella conica Persoon mycelium

Morchella conica Pers. strains of the study were isolated from fruit bodies collected in ash-mixed forests. At first, the strains were cultured on potato dextrose agar (PDA), then on modified Murashige and Skoog (MS) solid agar media. A normal-growing strain was chosen for the trehalase induction experiments. During the trehalase induction treatment, mycelia were grown in liquid culture containing different concentrations of trehalose. After the induction period of trehalase enzymes, physiological state of the mycelium and the oxidative stress were monitored in the vegetative mycelia by measuring the change of the malondialdehyde content, superoxide dismutase enzyme activity, the fresh and dry weight. The examined Morchella conica strain utilized the trehalose properly. The rising amount of the trehalose triggered the increase of the mycelial trehalase enzyme activity. Our results clearly proved that both neutral and acidic trehalase isoenzyme activity of the Morchella conica mycelium are inducible and are playing important role in the utilization of external trehalose.

A case study on cryopreservation of African sheep semen for the Red Maasai, Dorper breeds and their crosses

The objective of this study was to examine the effect of different concentrations of trehalose and raffinose in Tris-citric acid-glucose (TCG) egg yolk extender for the sperm freezability of Red Maasai sheep, another African breed- the Dorper and their crosses. The results showed that Red Maasai, Dorper and their crosses sperm could be cryopreserved by extending with 0.075 to 0.01 M trehalose or raffinose-TCG egg yolk extender. We conclude that the cryopreservation of currently under threat Red Maasai sheep due to uncontrolled exotic breeds sperm makes it possible to establish a gene bank for the storage of animal genetic resources for endangered species, under the management of the International Livestock Research Institute for future genetic options in an uncertain world. Key words: Red Maasai sheep sperm, freezability, raffinose, trehalose.

Effect of sugars on characteristics of Boer goat semen after cryopreservation

In order to improve Boer goat semen quality during cryopreservation process, the influence of sugar supplementation on semen characteristics of sperm were investigated. Three experiments were carried out to investigate the effect of (a) addition of two monosaccharides (fructose and glucose) and two disaccharides sugars (trehalose and sucrose) (b) sugar combination (fructose and trehalose, sucrose and trehalose, glucose and trehalose), and control (glucose without trehalose) (c) different concentrations of trehalose on cryopreservation using Tris based extender. The total motility, forward motility, viability, normal spermatozoa, acrosome integrity and membrane integrity were assessed subjectively. Differences were not detected among monosaccharides, but glucose increased ( P < 0.05) sperm forward motility in post-thaw goat semen compared to trehalose or sucrose supplementation. Semen quality did not differ ( P > 0.05) among disaccharide sugar supplementation. Combination of glucose and trehalose significantly improved the characteristics of Boer spermatozoa after cryopreservation ( P < 0.05). Supplementation of trehalose (198.24 mM) into the glucose extender significantly increased total motility, forward motility, live spermatozoa, acrosome integrity and membrane integrity following cryopreservation ( P < 0.05). In conclusion, glucose had the better ability to support Boer sperm motility and movement patterns. Combination of monosaccharide (glucose) and disaccharide (trehalose) improved semen quality following cryopreservation. Trehalose supplementation at the concentration of 198.24 mM to the glucose extender conferred the greater improvement of semen quality for Boer semen cryopreservation.

Thermal stability of α-amylase in aqueous cosolvent systems

The activity and thermal stability of alpha-amylase were studied in the presence of different concentrations of trehalose, sorbitol, sucrose and glycerol. The optimum temperature of the enzyme was found to be 50 +/- 2 degrees C. Further increase in temperature resulted in irreversible thermal inactivation of the enzyme. In the presence of cosolvents, the rate of thermal inactivation was found to be significantly reduced. The apparent thermal denaturation temperature (Tm) app and activation energy (Ea) of alpha-amylase were found to be significantly increased in the presence of cosolvents in a concentration-dependent manner. In the presence of 40% trehalose, sorbitol, sucrose and glycerol, increments in the (Tm)app were 20 degrees C, 14 degrees C, 13 degrees C and 9 degrees C, respectively. The Ea of thermal denaturation of alpha-amylase in the presence of 20% (w/v) trehalose, sorbitol, sucrose and glycerol was found to be 126, 95, 90 and 43 kcal/mol compared with a control value of 40 kcal/mol. Intrinsic and 8-anilinonaphathalene-1-sulphonic acid (ANS) fluorescence studies indicated that thermal denaturation of the enzyme was accompanied by exposure of the hydrophobic cluster on the protein surface. Preferential interaction parameters indicated extensive hydration of the enzyme in the presence of cosolvents.

Sugar feedingviatrehalose haemolymph concentration affects sex pheromone production in matedHeliothis virescensmoths

Long-distance, female-produced sex pheromones are widespread among moths. Larval feeding provides most of the nutrients for development of these insects but is not thought to influence the de novo production of the fatty-acid derived compounds used as pheromones by most species. Feeding on plant nectar (sugar) by adult moths is important for increasing female fitness and also for the pollination of many plant species. In this paper, I show that feeding on sucrose solution, as opposed to water, increases sex pheromone titre in mated, but not virgin, female Heliothis virescens. Mating caused a rapid decrease in haemolymph trehalose concentration, which was restored to near-virgin levels by sugar ingestion. When isolated mated female abdomens were cultured with different concentrations of trehalose, pheromone titre increased with increasing trehalose concentration. This effect was not observed when abdomens were cultured on saline containing the sugar rhamnose, which insects cannot metabolise to glucose. Virgins injected with the juvenile hormone (JH) analogue, methoprene, showed the same effects as mated females with respect to pheromone titre and haemolymph trehalose concentration. Thus, following mating increases in JH titre increase demand for, and lowering of, blood sugar to develop oocytes, which can be compensated for by sugar ingestion. Haemolymph trehalose concentration probably influences glycolysis in gland cells and, consequently, levels of cytosolic citrate and acetyl-CoA for pheromone biosynthesis. This increase in pheromone titre in sugar-fed, mated females may facilitate further mating and increased fecundity. Thus, exogenous sugar feeding is behaviourally and physiologically integrated with endogenous JH titre to maximise female fitness.

Trehalose inhibits the release of adipokinetic hormones from the corpus cardiacum in the African migratory locust, Locusta migratoria, at the level of the adipokinetic cells.

The effect of trehalose at various concentrations on the release of adipokinetic hormones (AKHs) from the adipokinetic cells in the glandular part of the corpus cardiacum of Locusta migratoria was studied in vitro. Pools of five corpora cardiaca or pools of five glandular parts of corpora cardiaca were incubated in a medium containing different concentrations of trehalose in the absence or presence of AKH-release-inducing agents. It was demonstrated that trehalose inhibits spontaneous release of AKH I in a dose-dependent manner. At a concentration of 80 mM, which is the concentration found in the hemolymph at rest, trehalose significantly decreased the release of AKH I induced by 100 microM locustatachykinin 1, 10 microM 3-isobutyl-1-methylxanthine (IBMX) or high potassium concentrations. The specificity of the effect of trehalose was studied by incubating pools of corpora cardiaca with the non-hydrolyzable disaccharide sucrose or with glucose, the degradation product of trehalose, both in the presence and absence of 10 microM IBMX. Sucrose had no effect at all on the release of AKH I, whereas glucose strongly inhibited its release. The results point to the inhibitory effect of trehalose on the release of AKH I being exerted, at least partly, at the level of the adipokinetic cells, possibly after its conversion into glucose. The data presented in this study support the hypothesis that in vivo the relatively high concentration of trehalose (80 mM) at rest strongly inhibits the release of AKHs. At the onset of flight, the demand for energy substrates exceeds the amount of trehalose that can be mobilized from the fat body and consequently the trehalose concentration in the hemolymph decreases. This relieves the inhibitory effect of trehalose on the release of AKHs, which in turn mobilize lipids from the fat body.

Fertilization and Development of Frozen–Thawed Germinal Vesicle Bovine Oocytes by a One-Step Dilution Methodin Vitro

The objective of this study was to evaluatein vitrofertilization and cleavage rates of frozen–thawed bovine oocytes at the germinal vesicle (GV) stage. In mouse oocytes, spindle microtubule reorganization after GV breakdown is particularly sensitive to cold and readily damaged by exposure to low temperatures, the damage becoming apparent only at the time of the first mitotic division. The effects of various permeating cryoprotective agents [1.8Methylene glycol (EG), 1.3Methylene glycol monomethyl ether (EME), and 1.6M1,2-propanediol (PROH)] and different concentrations of trehalose (T) and polyvinylpyrrolidone (PVP) on post-thaw developmental capacity were examined. When bovine GV oocytes were frozen slowly in mixtures of 1.8MEG plus 5% PVP and 0.05MT, almost 80% developed to metaphase II; 22.2% degenerated afterin vitromaturation, and none of those that had been cryopreserved underwent parthenogenetic activation. The total fertilization rate was higher (P< 0.05) for oocytes frozen in a mixture of 1.8MEG plus 0.05MT or 0.1MT than in a mixture of 1.8MEG with or without 0.2MT; however, there was no difference in the number of normally fertilized or polyspermic oocytes that had been frozen in various cryoprotective solutions. No significant difference was observed in subsequent development using EG, EME, and PROH for GV oocytes. The addition of 0.05 or 0.1Mtrehalose to the freezing solution yielded significantly better cleavage and blastocyst rates than the solutions containing 0.2Mor no trehalose. For unfrozen controls, GV oocytes yielded significantly higher (P< 0.01) cleavage and blastocyst rates compared with frozen–thawed GV oocytes. It was found that 5% PVP had a beneficial effect compared with 10 or 20% concentrations for the development of blastocysts. Transfer of six blastocysts derived from frozen–thawed GV oocytes into three recipient heifers resulted in three pregnancies and the birth of one set of twins and one singleton calf.

Normal Calves Obtained after Direct Transfer of Vitrified Bovine Embryos Using Ethylene Glycol, Trehalose, and Polyvinylpyrrolidone

In the present study, IVF bovine embryos were vitrified using as the cryoprotectants, ethylene glycol plus trehalose plus the polymer, polyvinylpyrrolidone (PVP). In Experiment I, toxicity of the vitrification solution (VS) containing 20% PVP was tested in relation to temperature and exposure time. One hundred percent embryo development was observed with treatment at 5°C for 5 min, whereas only 55.5% embryos were developed when the treatment was carried out at 20°C for 5 min. In Experiment II, embryos were vitrified using one of the three treatments (Treatment A, 40% ethylene glycol (EG); treatment B, 40% EG + 11.3% trehalose; and treatment C, 40% EG + 11.3% trehalose + 20% PVP and rehydrations) was performed directly in mPBS. Highest development (84.1%) and hatching rate (68.2%) were obtained when embryos were vitrified with the vitrification solution used in treatment C. In Experiment III, embryos were vitrified as in Experiment II (treatment C). The development and hatching rates were compared after rehydration in different rehydration solutions. No significant difference was observed among the development and hatching rates when rehydration was carried out in different concentrations of trehalose. Five vitrified–warmed bovine embryos were transferred directly to five recipients and three recipients gave birth to three normal calves.

Properties of gel phase lipid-trehalose bilayers upon rehydration

When dipalmitoylphosphatidylcholine bilayers dried under vacuum in different concentrations of trehalose are rehydrated in buffer without the sugar they show different physicochemical properties in the gel state in comparison to the normal gel state. Dry DPPC/trehalose mixtures are readily dispersed in buffer below the phase transition showing by electron microscopy a morphology similar to liposomes prepared by dispersing the lipids in buffer above the phase transition temperature. In these conditions, an increase in the peak at 570 nm of merocyanine after the dehydration-rehydration process in the presence of the sugar is observed and the water permeation increases to values comparable to those found in the fluid state as indicated by the activation energy values and the osmotic volume. The trehalose-dried liposomes rehydrated in buffer show a similar osmotic response to hypertonic gradient as DPPC liposomes without sugar near the phase transition temperature. In accordance with this behavior the trehalose-dried liposomes are lysed below the phase transition temperature by lysoderivatives. These modifications of the gel state of hydrated phospholipids by trehalose can only be achieved if a drastic dehydration is performed in the presence of the sugar. After rehydration the changes in the gel state can be detected after dyalizing the rehydrated membranes in media without trehalose during at least 24 h. These results suggest that trehalose is still intercalated between the phospholipids after restoring water to the dried liposomes either at temperatures below or above the phase transition.

Ultrarapid freezing of mouse embryos with DMSO and trehalose

In the present study, IVF bovine embryos were vitrified using as the cryoprotectants, ethylene glycol plus trehalose plus the polymer, polyvinylpyrrolidone (PVP). In Experiment I, toxicity of the vitrification solution (VS) containing 20% PVP was tested in relation to temperature and exposure time. One hundred percent embryo development was observed with treatment at 5°C for 5 min, whereas only 55.5% embryos were developed when the treatment was carried out at 20°C for 5 min. In Experiment II, embryos were vitrified using one of the three treatments (Treatment A, 40% ethylene glycol (EG); treatment B, 40% EG + 11.3% trehalose; and treatment C, 40% EG + 11.3% trehalose + 20% PVP and rehydrations) was performed directly in mPBS. Highest development (84.1%) and hatching rate (68.2%) were obtained when embryos were vitrified with the vitrification solution used in treatment C. In Experiment III, embryos were vitrified as in Experiment II (treatment C). The development and hatching rates were compared after rehydration in different rehydration solutions. No significant difference was observed among the development and hatching rates when rehydration was carried out in different concentrations of trehalose. Five vitrified–warmed bovine embryos were transferred directly to five recipients and three recipients gave birth to three normal calves.