Rooster spermatozoan motility,forward progression,and fertility after storage at 25, 5, or −196 °C in various extenders

Rooster spermatozoa were stored at 25, 5, or ?196 °C in either TC199, a pyruvate-lactate mouse ova culture medium, or as undiluted semen. There was a linear decrease in percent of motile sperm during storage at 25 or 5 °C in all cases, and a curvilinear decrease with increasing storage times at ?196 °C. Percent of motile sperm present after increasing storage time suggested pyruvate-lactate is a better extender than TC199 at the three storage temperatures studied. Pullets inseminated with 1 × 10⁸ motile sperm using fresh sperm diluted in TC199 or pyruvate-lactate, or stored 24 hr at 5 or ?196 °C produced 68.7, 74.1, 20.6, and 10.8% fertile eggs, respectively. The differences in fertility between controls or between samples stored at 5 and ?196 °C were not significant. However, fertility from sperm stored at 5 and ?196 °C was significantly lower (p < .05) than both control groups. Thus, it can be concluded that TC199 or pyruvate-lactate may be used to dilute fresh rooster semen collections prior to insemination. In contrast, fertility of rooster sperm is not satisfactorily maintained after 5 or ?196 °C storage for 24 hr in a pyruvate-lactate extender.     

Influence of incubation in seminal plasma on subsequent metabolic and morphological characteristics of bovine spermatozoa

Incubation of bovine spermatozoa at 25° C for up to 8 hours in seminal plasma had little influence on oxygen uptake as compared to preincubation values but increased both the number of dead spermatozoa and proportion of spermatozoa with detached acrosomes. Washing spermatozoa and resuspension in either saline or seminal plasma followed by incubation for 4 or 8 hours decreased oxygen uptake and at 8 hours decreased the proportion of cells with deteched acrosomes as compared to 8 hour control values. When spermatozoa were not washed but were extended in egg yolk citrate extender or seminal plasma, oxygen uptake was greater for cells extended with seminal plasma. Storage of spermatozoa for 14 days at ?196° C following incubation in seminal plasma showed little influence of incubation time in seminal plasma on postfreeze metabolic or morphological characteristics. These experiments indicate that incubation of spermatozoa in seminal plasma prior to storage has little beneficial effect and, on the contrary, may cause an increase in acrosomal loss.     

Mink semen studies: I. Liquid preservation and prospect of freezing spermatozoa

Using 22 males, 41 semen samples were collected from the vagina of mink by means of a plastic tubing attached to a 1 ml syringe. Subsamples of vaginal semen were diluted in 4 different extenders, viz., tris (tris, citric acid, glycine, fructose, glycerol and egg yolk), PVP (tris extender with polyvinyl pyrrolidone and caproic acid), milk (boiled and filtered milk with glycerol) and sodium citrate. The extended semen samples were stored at 23, 5 and −196°C for varying periods and evaluated for % motile spermatozoa. In the tris extender storage for 3 days at 5°C or for 2 days at 23°C reduced the number of spermatozoa by more than 50%. When milk was used as the extender, the motility decreased from an initial value of 68% to 10% after 5 days of 5°C and to 8% after 4 days at 23°C. The PVP extender was not suitable for storage at any temperature. After being frozen at −196°C for 2 hr, the motility ranged from 3–10% in the tris extender and was zero in milk and PVP extenders. Prolonged storage for 7 days in tris extender reduced the motility to 1–7%.     

Effect of addition of buserelin acetate to the extender on motility and viability of bovine spermatozoa

The present study was aimed to determine the effect of GnRH analog (buserelin acetate) on the quality of bovine spermatozoa stored at 16°?C for 24?h. Semen collected in the summer season from June to September from healthy Polish Holstein–Friesian bulls. Ejaculates were centrifuged, divided and diluted to the final concentration of 240?×?10⁶ spermatozoa/mL using animal protein–free commercial BIOXcell^® extender (IMV Technologies, L’aigle, France) (Control) or with BIOXcell^® extender supplemented with buserelin acetate and stored 0, 8 and 24?h. Sperm motility parameters analysis was performed using a computer-assisted sperm analysis (CASA) system. The viability of spermatozoa was performed using flow cytometer. The addition of buserelin acetate to BIOXcell^® extender did positively affect the total motility (was higher in the observed samples with the addition of 2?µg/mL and 4?µg/mL than in the control group), progressive motile (forward progressing sperm was significantly increased (p?<?0.05) over the control group at the 0?h and 8?h of incubation following the supplementation of 2, 4 and 8?μg/mL buserelin acetate) and viability of spermatozoa (the number of live spermatozoa was significantly higher (p?<?0.05) in 2?µg/mL and 4?µg/mL samples with buserelin acetate at 8th hour of incubation and in sample with 4?µg/mL at 24th hour of incubation compared to the control group). We recommend adding 4?µg/mL to the extender to improve the quality of bovine semen.     

On the nature of the oxygen uptake in the light by Chondrus crispus. Effects of inhibitors,temperature and light intensity

The nature of the different processes of O₂ uptake involved in the light in the red macroalga Chondrus crispus Stackhouse (Rhodophyta, Gigartinales) was investigated. At limiting CO₂, INH (2.5 mM) did not alter the O₂ uptake rate. Glycolate was not excreted and did not accumulate within the cells. KCN reduced the rate of O₂ uptake in the light by 76% at limiting CO₂ and by 43% at saturating CO₂, but caused > 95% inhibition of O₂ evolution. DCMU (5 μM) totally blocked the photosynthetic electron transport chain, but allowed a residual O₂ uptake of 3.0±0.6 μmol O₂ .h^?1.g^?1 FW, irrespective of the CO₂ concentration. In saturating CO₂, a high light intensity pretreatment significantly stimulated the rate of O₂ uptake compared to net O₂ evolution, suggesting the persistence, in the light, of mitochondrial respiration. Irrespective of the CO₂ concentration, the optimum temperature for O₂ evolution was 17°C whereas dark O₂ uptake increased linearly with temperature. In contrast, O₂ uptake in the light showed an optimum at 17°C in limiting CO₂, and 21–25° C in saturating CO₂; its Q₁₀ was 2.4 at limiting CO₂, a value close to that of RuBP oxygenase, and 3.1 at saturating CO₂, a value close to that of dark respiration. It is concluded that: 1) mitochondrial respiration and Mehler reaction are both involved at all CO₂ concentrations, 2) RuBP oxygenase activity cannot account for more than 45%, and Mehler reaction for less than 20%, of the total O₂ uptake observed in the light at limiting CO₂.     

The effects of development at sub-optimal growth temperatures on photosynthetic capacity and susceptibility to chilling-dependent photoinhibition in Zea mays

When plants of Zea mays L. cv. LG11 that have been grown at optimal temperatures are transferred to chilling temperatures (0–12°C) photoinhibition of photosynthetic CO₂ assimilation can occur. This study examines how growth at sub-optimal temperatures alters both photosynthetic capacity and resistance to chilling-dependent photoinhibition. Plants of Z. mays cv. LG11 were grown in controlled environments at 14, 17, 20 and 25°C. As a measure of the capacity for photosynthesis under light limiting conditions, the maximum quantum yields of CO₂ assimilation (φ^a.c) and O₂ evolution (φ^a.o) were determined for the laminae of the second leaves at photon fluxes of 50–150 μmol m^-2s^-1. To determine photosynthetic capacity at photon fluxes approaching light saturation, rates of CO₂ uptake (A₁₅₀₀) and O₂ evolution (A₁₅₀₀) were determined in a photon flux of 1500 μmol m^-2s^-1. In leaves developed at 14°C, φ and φ were 26 and 43%, respectively, of the values for leaves grown at 25°C. Leaves grown at 17°C showed intermediate reductions in φ and φ, whilst leaves developed at 20°C showed no significant differences from those grown at 25°C. Similar patterns of decrease were observed for A₁₅₀₀ and A_1500.0 with decreasing growth temperature. Leaves developed at 25°C showed higher rates of CO₂ assimilation at all light levels and measurement temperatures in comparison to leaves developed at 17 and 14°C. A greater reduction in A₁₅₀₀ relative to A_1500.0 with decreasing growth temperature was attributed to increased stomatal limitation. Exposure of leaves to 800–1000 μmol m^-2 s^-1 when plant temperature was depressed to ca 6.5°C produced a photoinhibition of photosynthetic CO₂ assimilation in all leaves. However, in leaves developed at 17°C the decrease in A₁₅₀₀ following this chilling treatment was only 25% compared to 90% in leaves developed at 25°C. Recovery following chilling was completed earlier in leaves developed at 17°C. The results suggest that growth at sub-optimal temperatures induces increased tolerance to exposure to high light at chilling temperatures. This is offset by the large loss in photosynthetic capacity imposed by leaf development at sub-optimal temperatures.     

Susceptibility of the cigarette beetle Lasioderma serricorne (Coleoptera: Anobiidae) to hypoxia

The susceptibility of the cigarette beetle Lasioderma serricorne (F.) to hypoxia was examined at three different oxygen concentrations (0.5?C0.8, 1.0?C1.3, and 2.0?C2.3?%) and four different temperature/humidity (RH) conditions: 30?°C/75?% RH, 25?°C/75?% RH, 20?°C/43?% RH, and 15?°C/43?% RH. The influence of humidity on mortality was also examined at three humidity levels (21, 43, and 75?% RH) at 1.0?C1.3?% oxygen (O₂) and 25?°C. Our results revealed that adult beetles were the most tolerant at 2.0?C2.3?% O₂ and that the larvae were the most tolerant at O₂ levels <1.0?C1.3?%. Mortality increased with increasing temperatures and decreasing O₂ concentrations. At 30?°C, 75?% RH, and 0.5?C0.8?% O₂, the 99?% lethality (LT₉₉) of larvae was 6.9?days; however, it increased to 20?days when the temperature was decreased to 25?°C or when O₂ levels were increased to 1.0?C1.3?%. Humidity also influenced mortality of both larval and adult beetles. LT₉₉ values for larvae at 25?°C and 1.0?C1.3?% O₂ were 24.0, 44.6, and 50.2?days at 21, 43, and 75?% RH, respectively. Results of this study indicate that a controlled atmosphere (CA) with reduced oxygen levels (<0.5?C0.8?% O₂) represents an effective measure for disinfesting stored tobacco as an alternative to conventional phosphine fumigation at temperatures >30?°C.     

Metabolism of the spade-headed Amphisbaenian worm lizard,Diplometopon zarudnyi (Nikolsky, 1907), in Saudi Arabia (Reptilia: Trogonophidae)

The oxygen consumption rate $\stackrel{?}{V}O2$ and lactate production of the Amphisbaenian worm lizard Diplometopon zarudnyi were measured at temperatures ranging from 15?°C to 35?°C at 5?°C intervals. The $\stackrel{?}{V}O2$ was significantly different between resting and active states at any specified temperature, while the average value at the resting state generally rose with increased temperature from 15?°C (0.05?ml O₂/g/h) to 25?°C (0.111?ml O₂/g/h). The aerobic respiration scopes at resting and active states were also significantly different. The highest Q10 values (3.24 and 1.69) were obtained at 15?°C–20?°C and 30?°C–35?°C during resting and active states, respectively, with these values being significantly different. Lactate concentrations were significantly higher during active states than when resting, and the anaerobic scope was found to increase with increased temperature. There was a proportional increase in ATP molecules (μmoles/g/2?min) during aerobic or anaerobic respiration, as well as in total metabolic scope, with increasing temperature, and the anaerobic scope showed significantly higher values than the aerobic scope, confirming the importance of anaerobic behavior for this species.     

Evidence for two forms of phospholipase A2 in human semen

The molecular weight of the active unit of phospholipase A₂ (PA₂) in human seminal plasma and spermatozoa was determined using the radiation inactivation technique. Fresh spermatozoa possess more than one form of PA₂ activity as judged by the biphasic nature of the curve obtained during enzyme inactivation. However, when stored frozen for several months followed by a period of heating for 60 min at 60 °C prior to irradiation, the sperm exhibited PA₂ activity, which corresponded to a single low molecular mass form of 12,000 d when radioactive phosphatidylcholine (PC) was used as substrate and 8,000 d when radioactive phosphatidylethanolamine (PE) was used as substrate. In fresh seminal fluid, only one active form of PA₂ was detected as judged by the linear nature of the curve obtained during enzyme inactivation by irradiation. Using PC as substrate, the active unit was again estimated to be 12,000 d, whereas it corresponded to 18,000 d when PE was used. The PA₂ activity associated with normal spermatozoa exhibited a 60% decrease in activity after storage at ?20 °C for 48 hr followed by a heating period of 10 min at 60 °C. Long-term storage of spermatozoa at ?20 °C also resulted in a similar decrease in the deacylation of PC. No further loss of activity was observed during subsequent heat treatment at 60 °C. Seminal plasma, however, showed no loss of activity following short (48 hr at 4 °C or ?20 °C) or long-term storage and subsequent heat treatment. Thus, the behavior of PA₂ when the effect of temperature was studied and in radiation inactivation experiments indicates that the low molecular weight component in the seminal plasma as well as in spermatozoa is temperature resistant. However, in fresh spermatozoa, a second form of PA₂ was found and was sensitive to changes in temperature.     

The effects of extenders and sperm‐egg ratios on fertilizing ability of cryopreserved testicular sperm of northern pike (Esox lucius L.)

The effects of four different extenders and two sperm‐to‐egg ratios on fertilizing ability of cryopreserved testicular sperm of northern pike (Esox lucius L.) were tested in the present study. Testicular sperm was diluted with each of the four extenders (0.6 m sucrose + 15% DMSO, 0.3 m glucose + 15% DMSO, 0.6 m sucrose + 10% methanol and 0.3 m glucose + 10% methanol, all supplemented with 10% egg yolk and 1.7 g KCl L^?1) and frozen in 0.5‐ml straws at 2 cm above the surface of liquid nitrogen and thawed at 25°C for 30 s. Then 125 μl or 50 μl of frozen‐thawed testicular sperm was poured onto about 1250 eggs for fertilization. The results showed that both sperm‐to‐egg ratio and diluent had no significant influence on cryopreservation efficiency of testicular sperm, whereas cryoprotectant had a significant influence. The highest fertilization rate (92.2%) was obtained from testicular sperm cryopreserved in glucose‐based extender containing 10% methanol at a sperm‐to‐egg ratio of 1 × 10⁶ spermatozoa per egg. The results indicated that glucose‐based extender containing 10% methanol, 10% egg yolk and 1.7 g KCl L^?1 was a useful combination.     

Characterization of phosphate absorption in maize root cortex segments

Uptake of phosphate ions by 1 mm segments of isolated maize root cortex layers was studied. Cortex segments (from roots of 8 days old maize plants) absorb phosphate ions from 1 mM KH₂PO₄ in 0.2 mM CaSCO₄ at the average rate of 34.3 ±3.2 μg P_i g^?1 (fr. m.) h^?1,i.e. 0.35± 0.02 μmol P_i g^?1 (fr. m.) h^?1. Phosphate uptake considerably increases after a certain period of “augmentation”,i.e. washing in aerated 0.2 mM CaSO₄. This increase is completely blocked by the presence of 10 μg ml^?1 cycloheximide. The relation of uptake rate to phosphate concentration in the medium was shown to have 3 phases in the concentration range of 0.02 - 40 mM. Transition points were found between 0.8–1 mM and 10–20 mM. Following K_m and V_max values were found: K_m[mM] : 0.37 - 3.82 - 27.67 V_max[μg P_i g^?1 (fr. m.) h^?1] : 3.33 - 39.40 - 66.67 We have found no sharp pH optimum for phosphate uptake. It proceeds at almost constant rate till pH 6.0 and then the uptake rate drops with increasing pH. At low phosphate concentrations (1 mM) the lowest uptake rate was found at 5 and 13 °C, while the uptake is higher at 5 °C than at 13 °C at phosphate concentrations higher than 1 mM. At these concentrations uptake rate at 35 °C is lower than at 25 °C. Phosphate uptake considerably decreased in anaerobic conditions. DNP and iodoacetate (0.1 mM) completely blocked phosphate uptake from 1 mM KH₂PO₄, while uptake from 5 and 10 mM KH₂PO₄ was left unaffected by these substances. The inhibitors of active - SH groups NEM and PCMB inhibited phosphate uptake: 10^?3 M NEM by 81.6%, 10⁴ M NEM by 42% and 10^?4 M PCMB by 42%.     

An EPR Investigation of Human Methaemoglobin Oxidation by Hydrogen Peroxide: Methods to Quantify all Paramagnetic Species Observed in the Reaction

The method of Electron Paramagnetic Resonance (EPR) spectroscopy was used to study the reaction of human methaemoglabin (metHb) with hydrogen peroxide. The samples for EPR measurements were rapidly frozen in liquid nitrogen at different times after H₂O₂ was added at 3- and 10-fold molar excess to 100 μM metHb in 50 mM phosphate buffer, pH 7.4, 37°C. Precautions were taken to remove all catalase from the haemoglobin preparation and no molecular oxygen evolution was detected during the reaction. On addition of H₂O₂ the EPR signals (- 196°C) of both high spin and low spin metHb rapidly decreased and free radicals were formed. The low temperature (- 196°C) EPR spectrum of the free radicals formed in the reaction has been deconvoluted into two individual EPR signals, one being an anisotropic signal (g° = 2.035 and g° = 2.0053), and the other an isotropic singlet (g = 2.0042, AH = 20 G). The former signal was assigned to peroxyl radicals. As the kinetic Pehaviour of both peroxyl (ROO^*) and nonperoxyl (P^*) free radicals were similar, we concluded that ROO^* radicals are not formed from P^* radicals by addition of O₂. The time courses for both radicals showed a steady state during the time required for H₂O₂ to decompose. Once all peroxide was consumed, the radical decayed with a first order rate constant of 1.42 ± 10^-3 s^-1 (1:3 molar ratio). The level of the steady state was higher and its duration shorter at lower initial concentration of H₂O₂. The formation of the rhombic Fe(III) non-haemcentres with g = 4.35 was found. Their yield was proportional to the H₂O₂ concentration used and the centers were ascribed to haem degradation products. The reaction was also monitored by EPR spectroscopy at room temperature. The kinetics of the free radicals measured in the reaction mixture at room temperature was similar to that observed when the fast freezing method and EPR measurement at —196°C were used.     

Influence of light and temperature on photosynthesis and respiration by Pithophora oedogonia (Mont.) Wittr. (chlorophyceae)

Rates of net photosynthesis and respiration were determined for Pithophora oedogonia (Mont.) Wittr. acclimatized to 56 combinations of light (7–1200 μE m^?2 s^?1) and temperature (5–35°C). Conditions for maximum net photosynthesis were estimated to be 26°C and 970 μE m^?2 s^?1. The rate of net photosyntheses varied considerably with temperature, with the maximum measured value (9.67 mg O₂ h^?1 g dry wt.^?1) occurring at 25°C. Respiration rate increased with temperature and the light received just prior to measurement. The maximum respiration rate (7.05 mg O₂ g^?1 h^?1) occurred at 30°C and 1200 μE m^?2 s^?1. Exposure of Pithophora to light levels of 600 or 1200 μE m^?2 s^?1 prior to determination of the respiration rate resulted in significantly elevated levels of oxygen consumption at temperatures ≥ 15°C. The relationship between light, temperature and photosynthesis and respiration were summarized as three-dimensional response surfaces.     

Fungicidal effect of hydrogen peroxide on fungal infection of rainbow trout eggs

Exposure to 1,500 μg/ml of hydrogen peroxide (H₂O₂) for 60 min at 13°C was found to be injurious to rainbow trout eggs. On the other hand, the concentration which effectively inhibited pathogenic fungi in vitro was substantially less than this toxic dosage; specifically, 500 μg/ml for 60 min at 20°C to inhibit the zoosporic stage and 1,000 μg/ml for 60 min at 20°C to inhibit the vegetative stage. From in vivo tests, treatment with 1,000 μg/ml of H₂O₂ for 60 min at 13°C was found to be the most effective procedure to control fungal infection and increase the hatching rate of rainbow trout eggs.     

Photosynthetic performance and resistance to photoinhibition of Zea mays L. leaves grown at sub-optimal temperature

The performance of the photosynthetic apparatus was examined in the third leaves of Zea mays L. seedlings grown at near-optimal (25 °C) or at suboptimal (15 °C) temperature by measuring chlorophyll (ChI) a fluorescence parameters and oxygen evolution in different temperature and light conditions. In leaf tissue grown at 25 and 15 °C, the quantum yield of PSII electron transport (ψ_PSII) and the rate of O₂ evolution decreased with decreasing temperature (from 25 to 4 °C) at a photon flux density of 125 μmol m^?2 s^?1. In leaves grown at 25 °C, the decrease of ψ_PSII correlated with a decrease of photochemical ChI fluorescence quenching (q_p), whereas in leaves crown at 15 °C q_p was largely insensitive to the temperature decrease. Compared with leaves grown at 25 °C, leaves grown at 15 °C were also able to maintain a higher fraction of oxidized to reduced Q_A (greater q_p) at high photon flux densities (up to 2000 μmol m^?2 s^?1), particularly when the measurements were performed at high temperature (25 °C). With decreasing temperature and/or increasing light intensity, leaves grown at 15 °C exhibited a substantial quenching of the dark level of fluorescence F₀ (q₀) whereas this type of quenching was virtually absent in leaves grown at 25 °C. Furthermore, leaves grown at 15 °C were able to recover faster from photo inhibition of photosynthesis after a photoinhibitory treatment (1200 μmol m^?2 s^?1 at 25, 15 or 6 °C for 8 h) than leaves grown at 25 °C. The results suggest that, in spite of having a low photosynthetic capacity, Z. mays leaves grown at sub optimal temperature possess efficient mechanisms of energy dissipation which enable them to cope better with photoinhibition than leaves grown at near-optimal temperature. It is suggested that the resistance of Z. mays leaves grown at 15 °C to photoinhibition is related to the higher content of carotenoids of the xanthophyll cycle (violaxanthin + antheraxanthin + zeaxanthin) measured in these leaves than in leaves grown at 25 °C.     

Bimodal oxygen uptake in a freshwater air-breathing fish,Notopterus chitala

Measurements of bimodal oxygen uptake have been made in a freshwater air-breathing fish,Notopterus chitala at 29.0±1(S.D.)°C. xhe mean oxygen uptake from continuously flowing water without any access to air, was found to be 3.58±0.37 (S.E.) ml O₂ · h^?1 and 56.84+4.29 (S.E.) ml O₂ · kg^?1 · h^?1 for a fish weighing 66.92 + 11.27 (S.E.) g body weight. In still water with access to air, the mean oxygen uptake through the gills were recorded to be 2.49 ± 0.31 (S.E.) ml O₂ · h^?1 and 38.78 ± 1.92 (S.E.) ml O₂ · kg^?1 · h^?1 and through the accessory respiratory organs (swim-bladder) 6.04±0.87 (S.E.) ml O₂ · h^?1 and 92.32±2.91 (S.E.) ml O₂ · kg^?1 · h^?1 for a fish averaging 66.92±11.27 (S.E.) g. Out of the total oxygen uptake (131.10 ml O₂ · kg^?1 · h^?1), about 70% was obtained through the aerial route and the remainder 30% through the gills.     

EFFECTS OF LIGHT AND TEMPERATURE ON NET PHOTOSYNTHESIS AND DARK RESPIRATION OF GAMETOPHYTES AND EMBRYONIC SPOROPHYTES OF MACROCYSTIS PYRIFERA1

The rates of net photosynthesis as a function of irradiance and temperature were determined for gametophytes and embryonic sporophytes of the kelp, Macrocystis pyrifera (L.) C. Ag. Gametophytes exhibited higher net photosynthetic rates based on oxygen and pH measurements than their derived embryonic sporophytes, but reached light saturation at comparable irradiance levels. The net photosynthesis of gametophytes reached a maximum of 66.4 mg O₂ g dry wt^?1 h^?1 (86.5 mg CO₂ g dry wt^?1 h^?1), a value approximately seven times the rate reported previously for the adult sporophyte blades. Gametophytes were light saturated at 70 μE m^?2 s^?1 and exhibited a significant decline in photosynthetic performance at irradiances 140 μE m^?1 s^?1. Embryonic sporophytes revealed a maximum photosynthetic capacity of 20.6 mg O₂ g dry wt^?1 h^?1 (25.3 mg CO₂ g dry wt^?1 h^?1), a rate about twice that reported for adult sporophyte blades. Embryonic sporophytes also became light saturated at 70 μE m^?2 s^?1, but unlike their parental gametophytes, failed to exhibit lesser photosynthetic rates at the highest irradiance levels studied; light compensation occurred at 2.8 μE m^?2 s^?1. Light-saturated net photosynthetic rates of gametophytes and embryonic sporophytes varied significantly with temperature. Gametophytes exhibited maximal photosynthesis at 15° to 20° C, whereas embryonic sporophytes maintained comparable rates between 10° and 20° C. Both gametophytes and embryonic sporophytes declined in photosynthetic capacity at 30° C. Dark respiration of gametophytes was uniform from 10° to 25° C, but increased six-fold at 30° C; the rates for embryonic sporophytes were comparable over the entire range of temperatures examined. The broader light and temperature tolerances of the embryonic sporophytes suggest that this stage in the life history of M. pyrifera is well suited for the subtidal benthic environment and for the conditions in the upper levels of the water column.     

Effects of Divalent Cations,Trypsin, and Phospholipases on the Passive Permeability to Sodium of Inside-Out Vesicles From Human Red Cells

Inside-out vesicles (IOV) were prepared from human red blood cells. Steady-state uptake of ²²Na was observed to generally follow an exponential time course with a rate constant of 1.57 ± 0.09 h^?1 (SE). One week of cold storage (0–4°C) increased the rate constant to 2.50 ± 0.12 h ^?1 (SE). Mg²⁺, Ca²⁺, or Sr²⁺ decreased the rate of ²²Na uptake with no observable differences between the three divalent cations when tested at concentrations of 50 μM. Mg²⁺ was shown to decrease the rate of ²²Na uptake at concentrations as low as 5 μM with maximal effect at 50 to 100 μM. The decrease in rate of ²²Na uptake induced by Mg²⁺ could be enhanced by exposure of IOV to Mg²⁺ for longer periods of time. Trypsin treatment of IOV increased the rate of uptake of ²²Na and was dependent on the concentration of trypsin added between 5 to 25 μg/ml (treated for 5 min at 25°C). The ability of Mg²⁺ (50 μM) to decrease the rate of ²²Na uptake was still observed after maximal trypsin treatment. Phospholipase A₂ or phospholipase C treatment of IOV increased the rate of ²²Na uptake and was dependent on the amount of phospholipase A₂ (0.1 to 1.0 units/ml) or phospholipase C (0.25 to 2.5 units/ml) added (treated for 5 min at 25°C). After phospholipase A₂ treatment, the observed decrease in the rate of ²²Na uptake induced by Mg²⁺ (50 μM) was generally greater than controls. After phospholipase C treatment, the observed decrease in rate of ²²Na uptake induced by Mg²⁺ (50 μM) was less or absent when compared with controls. Phospholipase C treatment was less effective in preventing the Mg²⁺ effect the longer IOV were exposed to Mg²⁺. The results suggest that Mg²⁺ binds to phospholipid head-groups to reduce Na permeability perhaps by inducing a change in bilayer structure or phospholipid association.     

Melatonin protects rabbit spermatozoa from cryo-damage via decreasing oxidative stress

Mammalian spermatozoa are highly susceptible to reactive oxygen species (ROS) stress. The aim of the present study was to investigate whether and how melatonin protects rabbit spermatozoa against ROS stress during cryopreservation. Semen was diluted with Tris-citrate-glucose extender in presence of different concentrations of melatonin. It was observed that addition of 0.1 mM melatonin significantly improved spermatozoa motility, membrane integrity, acrosome integrity, mitochondrial membrane potential as well as AMP-activated protein kinase (AMPK) phosphorylation. Meanwhile, the lipid peroxidation (LPO), ROS levels and apoptosis of post-thaw spermatozoa were reduced in presence of melatonin. Interestingly, when fresh spermatozoa were incubated with 100 μM H₂O₂, addition of 0.1 mM melatonin significantly decreased the oxidative damage compared to the H₂O₂ treatment, whereas addition of luzindole, an MT1 receptor inhibitor, decrease the effect of melatonin in spermatozoa. It was observed that the glutathione (GSH) content and activities of glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) were significantly increased with addition of melatonin during cryopreservation. In conclusion, addition of melatonin to the freezing extender protects rabbit spermatozoa against ROS attack by enhancing AMPK phosphorylation for increasing the antioxidative defense.     

The kinetics of light-induced changes of C-550, cytochrome b559 and fluorescence yield in chloroplasts at low temperature

The kinetics of the photoreduction of C-550, the photooxidation of cytochrome b₅₅₉ and the fluorescence yield changes during irradiation of chloroplasts at ?196 °C were measured and compared. The photoreduction of C-550 proceeded more rapidly than the photooxidation of cytochrome b₅₅₉ and the fluorescence yield increase followed the cytochrome b₅₅₉ oxidation. These results suggest that fluorescence yield under these conditions indicates the dark reduction of the primary electron donor to Photosystem II, P680⁺, by cytochrome b₅₅₉ rather than the photoreduction of the primary electron acceptor.The photoreduction of C-550 showed little if any temperature dependence over the range of ?196 to ?100 °C. The amount of cytochrome b₅₅₉ photooxidized was sensitive to temperature decreasing from the maximal change at temperatures between ?196 to ?160 °C to no change at ?100 °C. To the extent that the reaction occurred at temperatures between ?160 and ?100 °C the rate was largely independent of temperature. The rate of the fluorescence increase was dependent on temperature over this range being 3–4 times more rapid at ?100 than at ?160 °C. At ?100 °C the light-induced fluorescence increase and the photoreduction of C-550 show similar kinetics. The temperature dependence of the fluorescence induction curve is attributed to the temperature dependence of the dark reduction of P680⁺.The intensity dependence of the photoreduction of C-550 and of the photooxidation of cytochrome b₅₅₉ are linear at low intensities (below 200 μW/cm²) but fall off at higher intensities. The failure of reciprocity in the photoreduction of C-550 at the higher intensities is not explained by the simple model proposed for the Photosystem II reaction centers.