Vascular fluid shifts and endocrine responses to exercise in the heat. Effect of rehydration

This study examines the relationships between vascular changes and endocrine responses to prolonged exercise in the heat, associated with dehydration and rehydration by fluids of different osmolarity. Five subjects were exposed, in a 34 degrees C environment for 4 h of intermittent exercise on a cycle ergometer at 85 +/- 12 Watts (SD). Fluid regulatory hormones and cortisol were analysed in 3 experimental sessions: one without any fluid supplement (NO FLUID), and two with progressive rehydration, either by spring water (WATER) or isotonic solution (ISO), given after 70 min of exercise. Results were expressed in terms of differences between the mean values observed at the end of the exercise and the first hour values taken as references. Dehydration (NO FLUID) elicited a 4.0 +/- 0.8% (SE) decrease in plasma volume (PV) and an increase in osmolarity (8.4 +/- 3.1 mosmol X l-1). Concomitantly, plasma aldosterone (PA), renin activity (PRA), arginin vasopressin (AVP) and cortisol (PC) levels increased greatly in response to exercise in the heat (PA: 37.2 +/- 10.8 ng. 100 ml-1; PRA: 13.4 +/- 2.5 ng X ml-1 X h-1; AVP: 3.8 +/- 1.3 pg X ml-1; PC: 12.2 +/- 2.7 micrograms X 100 ml-1). Rehydration with water led to decreased osmolarity (-8.2 +/- 2.1 mosmol X l-1) with no significant changes in PV. With ISO, PV increased by 6.0 +/- 1.3% and the decrease in osmolarity was-5.8 +/- 1.8 mosmol X l-1. With both modes of rehydration, the increases in PRA, AVP and cortisol were blunted; only ISO prevented the rise in PA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Motility and cryopreservation of spermatozoa of European common frog, Rana temporaria

Motility and cryopreservation of testicular sperm of European common frog, Rana temporaria were investigated. Collected testicular spermatozoa were immotile in solutions of high osmolalities: 300 mmol/l sucrose and motility inhibiting saline solution-MIS. Full sperm motility could be activated in distilled water or in a solution of 50 mmol/l NaCl, = 90 mosmol/kg, with 75-90% motility and 14-16 μm s⁻¹ swimming velocity. Spermatozoa activated in distilled water and kept at room temperature ceased the motility within a period of 1 h. But when they were kept at 4 °C, no significant decrease in sperm motility and velocity occurred over a period of 1 h. Incubation of testicular sperm diluted 1:2 with MIS containing 10% DMSO, 5% glycerol, 10% methanol, or 10% propandiol for a period of 40 min at 4 °C showed that propandiol was the most toxic cryoprotectant for spermatozoa of European common frog R. temporaria. However, methanol was not toxic to spermatozoa during the 40 min incubation period, it failed to protect spermatozoa during the freezing and thawing process. DMSO and glycerol were useful penetrating cryoprotectants that interacted with sperm diluents in cryodiluent efficacy. In combination with the sucrose diluent, DMSO was a better cryoprotectant than glycerol, while in combination with MIS, DMSO and glycerol were similarly useful. Sperm was frozen at two freezing levels above the surface of liquid nitrogen. Sperm frozen 5 cm above the surface of liquid nitrogen resulted in immotile and non-viable spermatozoa. However, sperm frozen at 10 cm above the surface of liquid nitrogen showed 40-45% viability and 30-35% motility, compared to the untreated freshly collected testicular sperm. Addition of hen egg yolk had no positive effect on the post-thaw sperm motility, viability and hatching rate when added to sucrose cryodiluents. However, addition of 5% egg yolk to the MIS containing 5% glycerol and 2.5% sucrose significantly improved the hatching rate than all other treatments. Therefore, we conclude that, MIS and 300 mmol/l sucrose are suitable diluents for immotile storage of testicular semen. For cryopreservation, dilution to a final concentration of 5-6 × 10⁶/ml in MIS with 5% glycerol, 2.5% sucrose and 5% egg yolk, frozen in liquid nitrogen vapour at 10 cm above its surface, and thawed at 22 °C for 40 s is a useful cryopreservation protocol for R. temporaria sperm. Further research is needed to determine the motility parameters and cryopreservation of spermatic urine of R. temporaria.     

The influence of various cryoprotectants on semen quality of the rainbow trout (Oncorhynchus mykiss) before and after cryopreservation

The influence of permeating and of non-permeating cryoprotectants on motility, eosin permeability (sperm cells viability test) and leaking lactate dehydrogenase activity of spermatozoa of the rainbow trout ( Oncorhynchus mykiss ) was investigated. Correlations between sperm motility rate, percentage of eosinimpermeable (viable) cells and LDH activities established the three parameters as indicators for damages to rainbow trout spermatozoa. High sperm motility rates, velocities, and numbers of linear swimming sperm cells, high numbers of eosinimpermeable spermatozoa and a low extracellular LDH activity were evaluated as positive quality characteristics of semen. With a buffered physiological saline solution as basic extender we found a mixture of 0.67 mol L (5%) dimethylsulfoxide (DMSO) and 0.13 mol/L (1%) glycerol to be the most effective cryoprotectant, followed by DMSO (0.67 mol/L [5%]-1.85 mol/L [15%]) and glycerol (0.65 mol/L [5%]-1.78 mol/L [15%]) and propanediol (1.24mol L [10%]-1.78 mol/L [15%]). Addition of hen egg yolk (7%, 20%) and of 15 mmol/L (0.5%) sucrose significantly increased the semen quality in comparison to the same extender without these additives. Bovine serum albumin (1.5%, 3%) had no influence on the investigated semen parameters.     

Sucrose dilution of frozen mouse embryos: interaction of glycerol and sucrose concentrations

Several concentrations of glycerol for cryoprotection and several concentrations of sucrose for cryoprotectant dilution were examined with frozen, thawed and cultured mouse embryos. Four hundred and eighty late morulae to early blastocyst stage embryos were collected from 35 superovulated mice (B6D2 x Swiss Webster crosses back-crossed to Swiss Webster males) 3-1/2 days after breeding. The embryos were transferred through increasing concentrations of glycerol in modified Dulbecco(1)s phosphate buffered saline (MDPBS) to reach three final concentrations of 1.0 M, 1.4 M and 1.8 M. The embryos were loaded in 0.5-ml French straws appropriately filled with the cryoprotectant and sucrose solutions for each treatment. The straws were cooled with a standard fast-freezing program to -35 degrees C, then plunged into liquid nitrogen. After 58 days of storage at -196 degrees C the straws were thawed in a 37 degrees C water bath. Cryoprotectant dilution was accomplished with a standard step-wise procedure or in the straw with one of three concentrations of sucrose solution (0.25 M, 0.5 M, 1.0 M) in MDPBS. The embryos were then washed twice in MDPBS, twice in Whitten's media for embryo culture and then placed in microdrops of Whitten's media under paraffin oil in a water saturated 5% CO(2) in air atmosphere at 37 degrees C. Embryos were observed 24 hours later for development to the expanded blastocyst stage. The proportion of embryos developing in vitro from the three glycerol concentrations were not significantly different with standard step-wise dilution procedures for glycerol removal. After step-wise cryoprotectant removal, blastocyst expansion occurred in 49%, 44% and 52% of embryos frozen in 1.0 M, 1.4 M and 1.8 M glycerol, respectively. The 1.0 M sucrose dilution of 1.0 M glycerol showed the highest development (60.5%) in vitro but was not significantly different from any of these three step-wise diluted glycerol concentrations. The step-wise dilution of the three glycerol concentrations and dilution of the 1.0 M glycerol and 1.0 M sucrose were all superior (P < 0.01) to any other dilution procedure examined.     

Enhancement of the biocontrol agent Candida oleophila (strain O) survival and control efficiency under extreme conditions of water activity and relative humidity

The objective of this work is to evaluate the ability of some additive substances in protecting the biocontrol agent Candida oleophila (strain O) against the adverse effects of environmental factors, such as water activity (a_w, 0.93 and 0.98) and relative humidity (75% and 98%). The protection obtained with various protectant substances, skimmed milk (SM), peptone, maltose, sucrose, sorbitol, lactose and polyethylene glycol was assayed under in vitro and in vivo conditions. The yeast cells with the highest level of protecting agents (1%) had higher viability than those with low protectant levels (0.1% and 0.5%). SM, sucrose and sorbitol improved significantly the C. oleophila survival on apple fruit surface by 80.8%, 42.26% and 37.27% and gave a significant protection (from 96% to 100%) against Penicillium expansum under dried conditions. The highest strain O density and efficacy was obtained with SM. Under experimental conditions reflecting practical conditions, SM applied in combination with the strain O resulted in improved biocontrol efficacy by 74.65%. Therefore, SM could be used as material substrate with the best sugar protectants during the formulation process of this antagonistic yeast for eventual pre-harvest application.     

Culture-induced changes in osmolality of tobacco cell suspensions using four exogenous sugars

Suspension cultures of tobacco cells were grown in B5 media supplemented with sucrose, glucose, mannitol and sorbitol as exogenous sugars to examine culture-induced changes in the osmolality of the medium. Osmolality decreases were greatest in sucrose and glucose media during the 14 days in culture, and in glucose media were essentially linear, presumably reflecting the use of this sugar as a food source. Osmolality decreases occurred during the first week of culture in mannitol- and sorbitol-supplemented media, but later stabilized. Fresh weight of cultured cells in sucrose- and glucose-supplemented media increased by <200% during 14 days in culture, whereas cultured cells in mannitol- and sorbitol-supplemented media increased by only 39 and 48%, respectively. Cells transferred to the original liquid medium (B5 medium with 3% sucrose and 3% glucose) grew vigorously if they had been cultured in sucrose- and glucose-supplemented media; however, cells grown in mannitol- and sorbitol-supplemented media needed to be subcultured several times to recover their normal growth rate. By subculturing cells into increasingly higher conditions of sugars, cells tolerant to 560 mOsmol kg-1 H2O were obtained. The high osmolality-adapted cells increased by 140% in fresh weight in 8% glucose-supplemented medium. Glucose was best suited for producing the high osmolality required because sucrose concentrations at 10% sucrose and above resulted in cell death. To limit the decrease of osmolality in these suspension cultures requires changing the medium every 3 days to maintain osmolality above the 530 mOsmol kg-1 H2O needed to co-culture these as feeder cells with gametic and zygotic cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enhancement of the biocontrol agent Candida oleophila (strain O) survival and control efficiency under extreme conditions of water activity and relative humidity

The objective of this work is to evaluate the ability of some additive substances in protecting the biocontrol agent Candida oleophila (strain O) against the adverse effects of environmental factors, such as water activity (a_w, 0.93 and 0.98) and relative humidity (75% and 98%). The protection obtained with various protectant substances, skimmed milk (SM), peptone, maltose, sucrose, sorbitol, lactose and polyethylene glycol was assayed under in vitro and in vivo conditions. The yeast cells with the highest level of protecting agents (1%) had higher viability than those with low protectant levels (0.1% and 0.5%). SM, sucrose and sorbitol improved significantly the C. oleophila survival on apple fruit surface by 80.8%, 42.26% and 37.27% and gave a significant protection (from 96% to 100%) against Penicillium expansum under dried conditions. The highest strain O density and efficacy was obtained with SM. Under experimental conditions reflecting practical conditions, SM applied in combination with the strain O resulted in improved biocontrol efficacy by 74.65%. Therefore, SM could be used as material substrate with the best sugar protectants during the formulation process of this antagonistic yeast for eventual pre-harvest application.     

怀山药种质资源的包埋玻璃化超低温保存与植株再生

通过对怀山药（Dioscorea opposita）种质资源的包埋玻璃化超低温保存与植株再生进行研究,结果表明：将低温下锻炼7天的怀山药试管苗带芽茎段放入预培养基中,低温下培养3天,然后在室温下用3%的海藻酸钠和0.5mol·L-1CaCl2包埋,包埋珠用MS＋0.2mol·L-1蔗糖＋2mol·L-1甘油＋50g·L-1二甲基亚砜在0°C下装载60分钟,用30%甘油＋15%乙二醇＋10%二甲基亚砜＋15%聚乙二醇＋0.4mol·L-1蔗糖在0°C下脱水60分钟,迅速投入液氮,24小时后立即用40°C水浴快速化冻,再用MS＋0.5mol·L-1蔗糖溶液洗涤2次,转入再生培养基中培养,可获得再生植株。再生植株成活率因基因型而异,铁棍山药、太谷山药、怀庆1号山药和B号山药的成活率分别为64.29%、49.21%、13.11%和39.81%。     

The effect of citrate loading on exercise performance, acid-base balance and metabolism

Nine subjects (VO2max 65 +/- 2 ml.kg-1.min-1, mean +/- SEM) were studied on two occasions following ingestion of 500 ml solution containing either sodium citrate (C, 0.300 g.kg-1 body mass) or a sodium chloride placebo (P, 0.045 g.kg-1 body mass). Exercise began 60 min later and consisted of cycle ergometer exercise performed continuously for 20 min each at power outputs corresponding to 33% and 66% VO2max, followed by exercise to exhaustion at 95% VO2max. Pre-exercise arterialized-venous [H+] was lower in C (36.2 +/- 0.5 nmol.l-1; pH 7.44) than P (39.4 +/- 0.4 nmol.l-1; pH 7.40); the plasma [H+] remained lower and [HCO3-] remained higher in C than P throughout exercise and recovery. Exercise time to exhaustion at 95% VO2max was similar in C (310 +/- 69 s) and P (313 +/- 74 s). Cardiorespiratory variables (ventilation, VO2, VCO2, heart rate) measured during exercise were similar in the two conditions. The plasma [citrate] was higher in C at rest (C, 195 +/- 19 mumol.l-1; P, 81 +/- 7 mumol.l-1) and throughout exercise and recovery. The plasma [lactate] and [free fatty acid] were not affected by citrate loading but the plasma [glycerol] was lower during exercise in C than P. In conclusion, sodium citrate ingestion had an alkalinizing effect in the plasma but did not improve endurance time during exercise at 95% VO2max. Furthermore, citrate loading may have prevented the stimulation of lipolysis normally observed with exercise and prevented the stimulation of glycolysis in muscle normally observed in bicarbonate-induced alkalosis.     

Metabolic and circulatory responses to the ingestion of glucose polymer and glucose/electrolyte solutions during exercise in man

Six men exercised on a cycle ergometer for 60 min on two occasions one week apart, at 68 +/- 3% of VO2max. On one occasion, a dilute glucose/electrolyte solution (E: osmolality 310 mosmol X kg-1, glucose content 200 mmol X l-1) was given orally at a rate of 100 ml every 10 min, beginning immediately prior to exercise. On the other occasion, a glucose polymer solution (P: osmolality 630 mosmol X kg-1, glucose content equivalent to 916 mmol X l-1) was given at the same rate. Blood samples were obtained from a superficial forearm vein immediately prior to exercise and at 15-min intervals during exercise; further samples were obtained at 15-min intervals for 60 min at rest following exercise. Heart rate and rectal temperature were measured at 5-min intervals during exercise. Blood glucose concentration was not different between the two tests during exercise, but rose to a peak of 8.7 +/- 1.2 mmol X l-1 (mean +/- SD) at 30-min post-exercise when P was drunk. Blood glucose remained unchanged during and after exercise when E was drunk. Plasma insulin levels were unchanged during exercise and were the same on both trials, but again a sharp rise in plasma insulin concentration was seen after exercise when P was drunk. The rate of carbohydrate oxidation during exercise, as calculated from VO2 and the respiratory exchange ratio, was not different between the two tests.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leavening ability of baker's yeast exposed to hyperosmotic media

To develop a simple and rapid method for enhancing the leavening ability of baker's yeast, we examined the fermentation ability of baker's yeast exposed to hyperosmotic media. When baker's yeast cells were incubated at 25 degrees C for 1 h in a hyperosmotic medium containing 0.5% yeast extract, 0.5% peptone and 20% sucrose, the cells showed a higher fermentation ability in the subsequent fermentation test than those untreated. The increased ratios were from 40 to 60% depending on the strains used. Glucose and fructose showed a similar effect to that of sucrose, but sorbitol was less effective. A high correlation between the intracellular glycerol content and fermentation ability after the osmotic treatment suggested that glycerol accumulated during the hyperosmotic treatment was used in the subsequent fermentation as a substrate, lessened the lag time, and consequently enhanced the fermentation ability. Various baker's yeasts also showed a high leavening ability in dough after the hyperosmotic treatment.     

Osmotic stress as a factor in the detrimental effect of glycerol on human platelets

The aim of this work was to determine the importance of osmotic stress as a damaging factor in the detrimental effect of glycerol on human platelets. The severity of osmotic stress was mitigated by reducing the rate of change of glycerol concentration in the suspending medium. The classical permeability equations were used to predict cell volume changes in response to step changes in extracellular glycerol concentration. Protocols were devised that limited cellular shrinkage during glycerol addition and cellular swelling during glycerol dilution. When glycerol was added and diluted rapidly, the recovery of the hypotonic stress response with respect to untreated controls was unaffected by 0.25 mol/liter glycerol, but was reduced to ca. 65% after exposure to 0.5 mol/liter glycerol and to ca. 25% after exposure to 1 mol/liter glycerol. When 1 mol/liter glycerol was added and removed slowly such that cell volume remained within the range of 60-130% of normal volume, recovery of the hypotonic stress response was improved to ca. 50%, and the aggregation response was undiminished. Osmotic stress was therefore at least partly responsible for the damage caused by glycerol. However, platelets were damaged more after slow dilution from 1 mol/liter glycerol, when cellular swelling was limited to 116% of normal volume, than after rapid dilution from 0.25 or 0.5 mol/liter glycerol, which resulted in cellular swelling to 123% and 146% of normal volume, respectively. Thus, a possible toxic effect of glycerol cannot as yet be discounted.     

In situ detoxification and continuous cultivation of dilute-acid hydrolyzate to ethanol by encapsulated S. cerevisiae

Dilute-acid lignocellulosic hydrolyzate was successfully fermented to ethanol by encapsulated Saccharomyces cerevisiae at dilution rates up to 0.5h(-1). The hydrolyzate was so toxic that freely suspended yeast cells could ferment it continuously just up to dilution rate 0.1h(-1), where the cells lost 75% of their viability measured by colony forming unit (CFU). However, encapsulation increased their capacity for in situ detoxification of the hydrolyzate and protected the cells against the inhibitors present in the hydrolyzate. While the cells were encapsulated, they could successfully ferment the hydrolyzate at tested dilution rates 0.1-0.5h(-1), and keep more than 75% cell viability in the worst conditions. They produced ethanol with yield 0.44+/-0.01 g/g and specific productivity 0.14-0.17 g/(gh) at all dilution rates. Glycerol was the main by-product of the cultivations, which yielded 0.039-0.052 g/g. HMF present in the hydrolyzate was converted 48-71% by the encapsulated yeast, while furfural was totally converted at dilution rates 0.1 and 0.2h(-1) and partly at the higher rates. Continuous cultivation of encapsulated yeast was also investigated on glucose in synthetic medium up to dilution rate 1.0 h(-1). At this highest rate, ethanol and glycerol were also the major products with yields 0.43 and 0.076 g/g, respectively. The experiments lasted for 18-21 days, and no damage in the capsules was detected.     

Comparison of combinations of diluents and media for enumerating Zygosaccharomyces rouxii in intermediate water activity foods

Combinations of five diluents (0.1% peptone, 40 and 50% glucose, and 18 and 26% glycerol) and three enumeration media (tryptone glucose yeast extract, dichloran 18% glycerol and malt extract yeast extract 50% glucose (MY50G) agars) were evaluated for recovering a xerotolerant yeast, Zygosaccharomyces rouxii , from foods with intermediate water activity ( a _w). Combinations of 40% ( a _w, 0.936) or 50% ( a _w 0.898) glucose diluent and MY50G agar ( a _w 0.890) were superior in recovering highest populations. The type of solute in the diluent, as well as a reduced a _w, influences efficiency of recovering viable cells.     

Sucrose dilution of glycerol from mouse embryos frozen rapidly in liquid nitrogen vapour

The toxic effects of sucrose and the conditions of in-straw glycerol removal after freezing and thawing were studied using Day-3 mouse embryos. At 20 degrees C, exposure to less than or equal to 1.0 M-sucrose for periods up to 30 min had no adverse effects on freshly collected embryos. At 25 and 36 degrees C, however, greater than or equal to 1.0 M-sucrose significantly reduced the developmental potential (P less than 0.001). In the freezing experiments the embryos were placed in 0.5 ml straws containing 40 microliters freezing medium separated by an air bubble from 440 microliters sucrose solution. The straws were frozen rapidly in the vapour about 1 cm above the surface of liquid nitrogen. The post-thaw viability was substantially better after sucrose dilution at 20 degrees C than at 36 degrees C. Mixing the freezing medium with the sucrose diluent immediately after thawing further improved the rate of survival relative to mixing just before freezing (P less than 0.001). The best survival was obtained when the freezing medium contained 3.0 M-glycerol + 0.25 M-sucrose; it was mixed with the diluent after thawing and the glycerol was removed at 20 degrees C. Under such conditions the sucrose concentration in the diluent had no significant effect on the rate of development (0.5 M, 69%; 1.0 M, 73%; 1.5 M, 64%). The results show that during sucrose dilution the temperature should be strictly controlled and suggest that intracellular and extracellular concentrations of glycerol are important in the cryoprotection of embryos.     

Pre-exercise hypervolemia and cycle ergometer endurance in men

Time to exhaustion at 87-91% of peak VO2 was measured in 5 untrained men (age: 31 +/- 8 years, body mass: 74.20 +/- 16.50 kg, body surface area: 1.90 +/- 0.24 m2, peak VO2: 2.87 +/- 0.40 l min-1, plasma volume: 3.21 +/- 0.88 l; means +/-SD) after consuming nothing (N) or two fluid formulations (10 ml kg-1, 743 +/- 161 ml): Performance 1 (P1), a multi-ionic carbohydrate drink, containing 55 mEq l-1 Na+, 4.16 g l-1 citrate, 20.49 g l-1 glucose, and 365 mOsm kg-1 H2O, and AstroAde (AA), a sodium chloride-sodium citrate hyperhydration drink, containing 164 mEq l-1 Na+, 8.54 g l-1 citrate, <5 mg l-1 glucose, and 253 mOsm kg-1 H2O. Mean (+/-SE) endurance for N, P1 and AA was 24.68 +/- 1.50, 24.55 +/- 1.09, and 30.50 +/- 3.44 min respectively. Percent changes in plasma volume (PV) from -105 min of rest to zero min before exercise were -1.5 +/- 3.2% (N), 0.2 +/- 2.2% (P1), and 4.8 +/- 3.0% (AA; P < 0.05). The attenuated endurance for N and P1 could not be attributed to differences in exercise metabolism (VE, RE, VO2) from the carbohydrate or citrate, terminal heart rate, levels of perceived exertion, forehead or thigh skin blood flow velocity, changes or absolute termination levels of rectal temperature. Thus, the higher level of resting PV for AA just before exercise, as well as greater acid buffering and possible increased energy substrate from citrate, may have contributed to the greater endurance.     

In Vitro Divisions of Unfertilized Central Cells and Other Embryo Sac Cells in Nicotiana tabacum var macrophylla

The embryo sacs and female cells could be isolated from the unfertilized ovules of Nicotiana tabacum L. var. macrophylla which were treated in a solution containing 1.5 % cellulase R- 1O, 1% macerozyme R-10, 10% mannitol, 10 mmol/L CaCI:, pH 5.8 for 3 h followed by given slight pressure with a micropipette. The central cells could be kept viable for 10 h and the egg cells for 3 h in 10% mannital. Sometimes, the in situ fusion products of egg cell and synergid protoplasts could be obtained and kept viable for at least 5 h. The high concentration (20 mg/L) of 2, 4-D was used in enzyme solution to induce the division of the unfertilized central cells and other megagametophytic cells in subsequent culture. Treatment of 2,4-D together with enzymatic maceration of ovules was proved to be better than its direct treatment of isolated embryo sac or its component cells. Isolated embryo sacs were cultured in microchambers (Millicell-CM PICM 012 50 MILLIPORE) feeded with divided mesophyll protoplasts of Nicotiana rustica L. The medium was KMSp medium supple- mented with 1% glucose, 0.1 mol/L mannitol, 0.1 mol/L sorbitol, 0.25 mol/L sucrose, 1 mg/L BA, 6% to 10% coconut water, and 0.15% low gelling agarose. Division of central cells, antipodal cells and the in situ fusion products of egg cell and synergid protoplasts were induced. The unfertilized central cell was for the first time to be induced in vitro to develop into small cell clusters.     

Effect of polyols on the post-thawing motility of pellet-frozen ram spermatozoa

The cryoprotective effects of polyols in the absence and presence of glycerol in Tris-glucose-egg yolk based diluents on the post-thawing motility and acrosome integrity of pellet-frozen ram spermatozoa were examined. Incorporation of adonitol or xylitol (low molecular weight polyols; LMWPs) in diluents improved motility of spermatozoa in the absence of glycerol with maximum motility at 0.3 M (26.9 vs 13.3% mean motile spermatozoa; P < 0.001). Five concentrations of adonitol (0, 150, 300, 450, 600 mM) were examined in diluents containing 5 concentrations of glycerol (0, 1.5, 3.0, 4.5, 6.0% v/v). There was an additive effect of incorporation of 1.5% v/v glycerol with up to 450 mM adonitol (P < 0.001), but at higher levels of glycerol the incorporation of adonitol was detrimental to motility. The acrosome integrity of spermatozoa in diluents containing 0, 150 and 300 mM adonitol was superior to those containing 450 and 600 mM adonitol (46.1 vs 35.1% mean intact acrosomes; P < 0.05). Among the high molecular weight polyols (HMWPs) examined, better recovery of spermatozoa was obtained in diluents containing sorbitol than mannitol or inositol (P < 0.001). Sorbitol or mannitol (300 mM) improved the motility of spermatozoa in diluents without glycerol (P < 0.001), but the incorporation of HMWPs was detrimental in diluents containing glycerol. All five polyols were examined in isotonic diluents containing 360:0, 300:55, 240:110, 180:165, 120:220mM (Tris:polyol; 360 mosmol) and 6.0% v/v glycerol. There was a linear decrease in motility and acrosome integrity of spermatozoa with increasing polyol concentration in the diluent (P < 0.001) except for inositol, which was not detrimental. We conclude that the polyols examined have a cryoprotective effect on pellet-frozen ram spermatozoa except for inositol. However, in our study, no combination of polyols and glycerol was superior in terms of post-thawing motility and acrosome integrity of spermatozoa to 6.0% v/v glycerol alone in Tris-glucose-egg yolk diluents.     

Effect of sugars-addition on the survival of vitrified bovine blastocysts produced in vitro

We investigated the effect of addition of sugars to a vitrification solution on the survival rate of bovine blastocysts produced in vitro. In vitro-matured (IVM) and in vitro-fertilized (IVF) bovine Day-6 to Day-8 bovine blastocysts were classified into 3 developmental stages: early blastocysts, blastocysts and expanded blastocysts. The blastocysts were cryopreserved in 1 of 3 vitrification solutions: 1) 25% glycerol25% ethylene glycol (GE); 2) 20% glycerol20% ethylene glycol3/4 M sucrose (GES); and 3) 20% glycerol20% ethylene glycol3/8 M sucrose3/8 M dextrose (GESD). The basic solution was Dulbecco's PBS supplemented with 20% of fetal calf serum. Embryos were exposed to each vitrification solution in 3 steps, and after loading into 0.25-ml straws, were plunged into liquid nitrogen. After warming in water bath at 20 degrees C, cryoprotectants were diluted in 1/2 M and 1/4 M sucrose each for 5 min. Equilibration and dilution procedure except warming were conducted at room temperature (23 to 27 degrees C). After dilution, the embryos were cultured in Ham's F10 medium0.1 mM beta-mercaptoethanol20% fetal calf serum. Survival rates of embryos at 48 h of incubation of each of the 3 developmental stages (early blastocysts, blastocysts and expanded blastocysts) exposed to the 3 types of the vitrification solutions (GE, GES and GESD) were 23.5, 33.3, 65.8% (early blastocysts, blastocysts and expanded blastocysts respectively) in GE, 55.6, 71.9, 90.5% in GES and 84.6, 83.3, 95.8% in GESD respectively. These results indicate that a mixture of 25% glycerol25% ethylene glycol is not suitable for vitrification of early bovine blastocysts; however, addition of sugars to the solution significantly (P<0.01) improved the survival rate of the vitrified blastocysts, independently of their stage of development.     

Osmotic properties of the sealed tubular system of toad and rat skeletal muscle

A method was developed that allows conversion of changes in maximum Ca(2+)-dependent fluorescence of a fixed amount of fluo-3 into volume changes of the fluo-3-containing solution. This method was then applied to investigate by confocal microscopy the osmotic properties of the sealed tubular (t-) system of toad and rat mechanically skinned fibers in which a certain amount of fluo-3 was trapped. When the osmolality of the myoplasmic environment was altered by simple dilution or addition of sucrose within the range 190-638 mosmol kg(-1), the sealed t-system of toad fibers behaved almost like an ideal osmometer, changing its volume inverse proportionally to osmolality. However, increasing the osmolality above 638 to 2,550 mosmol kg(-1) caused hardly any change in t-system volume. In myoplasmic solutions made hypotonic to 128 mosmol kg(-1), a loss of Ca(2+) from the sealed t-system of toad fibers occurred, presumably through either stretch-activated cationic channels or store-operated Ca(2+) channels. In contrast to the behavior of the t-system in toad fibers, the volume of the sealed t-system of rat fibers changed little (by <20%) when the osmolality of the myoplasmic environment changed between 210 and 2,800 mosmol kg(-1). Results were also validated with calcein. Clear differences between rat and toad fibers were also found with respect to the t-system permeability for glycerol. Thus, glycerol equilibrated across the rat t-system within seconds to minutes, but was not equilibrated across the t-system of toad fibers even after 20 min. These results have broad implications for understanding osmotic properties of the t-system and reversible vacuolation in muscle fibers. Furthermore, we observed for the first time in mammalian fibers an orderly lateral shift of the t-system networks whereby t-tubule networks to the left of the Z-line crossover to become t-tubule networks to the right of the Z-line in the adjacent sarcomere (and vice versa). This orderly rearrangement can provide a pathway for longitudinal continuity of the t-system along the fiber axis.