Release of monomeric sugars from Miscanthus sinensis by microwave-assisted ammonia and phosphoric acid treatments

Microwave-assisted ammonium hydroxide (NH₄OH) followed by phosphoric acid (H₃PO₄) treatments were used to release monomeric sugars from Miscanthus sinensis grown in Cha-Chueng-Sao province, Thailand. Treatment with 1.0% (w/v) NH₄OH, 15:1 liquid-to-solid ratio (LSR) at 120 °C temperature for 15 min liberated 2.9 g of monomeric sugars per 100 g of dried biomass, whereas the corresponding yield for a treatment with 1.78% v/v H₃PO₄, 15:1 LSR at 140 °C for 30 min was 62.3 g/100 g. The two-stage pretreatment, treatment with NH₄OH at 120 °C temperature for 15 min followed by treatment with H₃PO₄ at 140 °C for 30 min, impressively provided the highest total monomeric sugar yield of 71.6 g/100 g dried biomass.     

Zinc chloride mediated degradation of cellulose at 200 °C and identification of the products

The effect of ZnCl₂ on the degradation of cellulose was studied to develop conditions to produce useful feedstock chemicals directly from cellulosic biomass. Cellulose containing 0.5 mol of ZnCl₂/mol of glucose unit of cellulose was found to degrade at 200 °C when heated for more than 60 s in air. The major non-gaseous products of the degradation were identified as furfural, 5-hydroxymethylfurfural and levulinic acid. The maximum yields for furfural and 5-hydroxymethylfurfural are 8% and 9%, respectively, based on glucose unit of cellulose. These yields are reached after 150 s of heating at 200 °C. A cellulose sample containing 0.5 mol of ZnCl₂/mol of glucose unit of cellulose and 5.6 equivalents of water when heated for 150 s at 200 °C produced levulinic acid as the only product in 6% yield. The ZnCl₂ mediated controlled degradation of cellulose at 200 °C is shown to produce useful feedstock chemicals in low yield.     

Dubiumin, a chymotrypsin-like serine protease from the seeds of Solanum dubium Fresen

A serine protease was purified 6.7-fold and with 35% recovery from the seeds Solanum dubium Fresen by a simple purification procedure that combined ammonium sulfate fractionation, cation exchange and gel filtration chromatographies. The enzyme, named dubiumin, has a molecular mass of 66 kDa as estimated by gel filtration and SDS-PAGE. Carbohydrate staining established the existence of a carbohydrate moiety attached to the enzyme. Inhibition of enzyme activity by serine protease inhibitors such as PMSF and chymostatin indicated that the enzyme belongs to the chymotrypsin-like serine protease class. Dubiumin is a basic protein with pI value of 9.3, acts optimally at pH 11.0, and is stable over a wide range of pH (3.0-12.0). The enzyme is also thermostable retaining complete activity at 60 °C after 1 h and acts optimally at 70 °C for 30 min. Furthermore, it is highly stable in the presence of various denaturants (2.0% SDS, 7.0 M urea and 3.0 M guanidine hydrochloride) and organic solvents [CH₃CN-H₂O (1:1, v/v) and MeOH-H₂O (1:1, v/v)] when incubated for 1 h. The enzyme showed a high resistance to autodigestion even at low concentrations.     

Hydrothermal synthesis and characterization of a new inorganic-organic hybrid compound AMP[ZnCl3] (AMP = 2-aminomethylpyridinium)

A novel organic-inorganic hybrid complex [(2-NH₂CH₂C₅H₄N)ZnCl₃] has been hydrothermally synthesized and characterized by single crystal X-ray diffraction, thermal analysis and spectroscopic studies. The compound crystallizes in the triclinic system, space group , a = 7.5339(9), b = 7.589(2), c = 9.365(2) Å, α = 104.55(2)°, β = 97.22(1)°, γ = 87.88(2)°, V = 513.6(2), Z = 2. In the title compound the 2-aminomethylpyridine acts as a ligand covalently linked to Zn(II) cation to form a slightly distorted ZnCl₃N tetrahedral environment. Each [Zn(C₆H₈N₂)Cl₃] unit is connected to one neighbor by a pair of hydrogen bonds between the apical chlorides and amine hydrogen atoms and to the other by a couple of π-π stacking interactions between the aromatic rings of the coordinated ligands forming a novel one-dimensional chain-like arrangement. The title complex is the first one that contains both coordinated and hydrogen bonded 2-aminomethylpyridine. Solid state ¹³C and ¹⁵N CP-MAS-NMR spectroscopies are in agreement with the X-ray structure. Ab initio calculations allow the attribution of carbons and nitrogen to the independent crystallographic sites. The Raman spectroscopy confirmed the presence of Zn-Cl and Zn-N bonds.     

Preparative separation of alkaloids from Nelumbo nucifera leaves by conventional and pH-zone-refining counter-current chromatography

Two modes of high-speed counter-current chromatography (HSCCC) were successfully applied to the separation of alkaloids from crude extract of Nelumbo nucifera leaves. The conventional HSCCC separations were performed with a two-phase solvent system composed of tetrachloromethane–CHCl₃–methanol–0.1 M HCl at a volume ratio of 1:3:3:2 (v/v/v/v), and 120 mg crude extract could be successfully separated. pH-Zone-refining CCC was performed with a two-phase solvent system composed of petroleum ether (60–90 °C)–ethyl acetate–methanol–water (5:5:2:8, v/v/v/v) where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluent. From 4.0 g of the crude extract, 120 mg N-nornuciferine, 1020 mg nuciferine and 96 mg roemerine were obtained in a single run each with a purity of over 98% as determined by HPLC. The structures of the isolated compounds were identified by ESI-MS, ¹H NMR and ¹³C NMR.     

Heat tolerance, behavioural temperature selection and temperature-dependent respiration in larval Octopus huttoni

This study reports temperature effects on paralarvae from a benthic octopus species, Octopus huttoni, found throughout New Zealand and temperate Australia. We quantified the thermal tolerance, thermal preference and temperature-dependent respiration rates in 1-5 days old paralarvae. Thermal stress (1 °C increase h⁻¹) and thermal selection (∼10-24 °C vertical gradient) experiments were conducted with paralarvae reared for 4 days at 16 °C. In addition, measurement of oxygen consumption at 10, 15, 20 and 25 °C was made for paralarvae aged 1, 4 and 5 days using microrespirometry. Onset of spasms, rigour (CT_max) and mortality (upper lethal limit) occurred for 50% of experimental animals at, respectively, 26.0±0.2 °C, 27.8±0.2 °C and 31.4±0.1 °C. The upper, 23.1±0.2 °C, and lower, 15.0±1.7 °C, temperatures actively avoided by paralarvae correspond with the temperature range over which normal behaviours were observed in the thermal stress experiments. Over the temperature range of 10 °C-25 °C, respiration rates, standardized for an individual larva, increased with age, from 54.0 to 165.2 nmol larvae⁻¹ h⁻¹ in one-day old larvae to 40.1-99.4 nmol h⁻¹ at five days. Older larvae showed a lesser response to increased temperature: the effect of increasing temperature from 20 to 25 °C (Q₁₀) on 5 days old larvae (Q₁₀=1.35) was lower when compared with the 1 day old larvae (Q₁₀=1.68). The lower Q₁₀ in older larvae may reflect age-related changes in metabolic processes or a greater scope of older larvae to respond to thermal stress such as by reducing activity. Collectively, our data indicate that temperatures >25 °C may be a critical temperature. Further studies on the population-level variation in thermal tolerance in this species are warranted to predict how continued increases in ocean temperature will limit O. huttoni at early larval stages across the range of this species.     

Evaluation of cryoprotectant and cooling rate for sperm cryopreservation in the euryhaline fish medaka Oryzias latipes

Medaka Oryzias latipes is a well-recognized biomedical fish model because of advantageous features such as small body size, transparency of embryos, and established techniques for gene knockout and modification. The goal of this study was to evaluate two critical factors, cryoprotectant and cooling rate, for sperm cryopreservation in 0.25-ml French straws. The objectives were to: (1) evaluate the acute toxicity of methanol, 2-methoxyethanol (ME), dimethyl sulfoxide (Me₂SO), N,N-dimethylacetamide (DMA), N,N-dimethyl formamide (DMF), and glycerol with concentrations of 5%, 10%, and 15% for 60 min of incubation at 4 °C; (2) evaluate cooling rates from 5 to 25 °C/min for freezing and their interaction with cryoprotectants, and (3) test fertility of thawed sperm cryopreserved with selected cryoprotectants and associated cooling rates. Evaluation of cryoprotectant toxicity showed that methanol and ME (5% and 10%) did not change the sperm motility after 30 min; Me₂SO, DMA, and DMF (10% and 15%) and glycerol (5%, 10% and 15%) significantly decreased the motility of sperm within 1 min after mixing. Based on these results, methanol and ME were selected as cryoprotectants (10%) to evaluate with different cooling rates (from 5 to 25 °C/min) and were compared to Me₂SO and DMF (10%) (based on their use as cryoprotectants in previous publications). Post-thaw motility was affected by cryoprotectant, cooling rate, and their interaction (P ? 0.000). The highest post-thaw motility (50 ± 10%) was observed at a cooling rate of 10 °C/min with methanol as cryoprotectant. Comparable post-thaw motility (37 ± 12%) was obtained at a cooling rate of 15 °C/min with ME as cryoprotectant. With DMF, post-thaw motility at all cooling rates was ?10% which was significantly lower than that of methanol and ME. With Me₂SO, post-thaw motilities were less than 1% at all cooling rates, and significantly lower compared to the other three cryoprotectants (P ? 0.000). When sperm from individual males were cryopreserved with 10% methanol at a cooling rate of 10 °C/min and 10% ME with a rate of 15 °C/min, no difference was found in post-thaw motility. Fertility testing of thawed sperm cryopreserved with 10% methanol at a rate of 10 °C/min showed average hatching of 70 ± 30% which was comparable to that of fresh sperm (86 ± 15%). Overall, this study established a baseline for high-throughput sperm cryopreservation of medaka provides an outline for protocol standardization and use of automated processing equipment in the future.     

Purification, characterization and mass spectroscopic analysis of thermo-alkalotolerant ��-1, 4 endoxylanase from Bacillus sp. and its potential for dye decolorization

A Bacillus sp., isolated from sludge and sediments of pulp and paper mill, was found to produce xylanase in a synthetic culture media containing oat spelt xylan (1% w/v) and 10% black liquor as inducers along with 2.5% (w/v) sucrose as additional carbon source. The purified enzyme was highly thermostable with half-life of 10 min at 90 °C and pH 8. The enzyme was stable over a broad range of pH (pH 6-10) and showed good thermal stability when incubated at 70 °C. Chemicals like EDTA, Hg²⁺, Cu²⁺ and solvents like glycerol and acetonitrile completely inhibited enzyme activity at high concentration. The molecular weights of the purified enzyme, determined by matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI-TOF/MS) analysis was analogous to the results obtained from SDS-PAGE, i.e. 55 kDa. Kinetic parameters were determined by using oat spelt xylan as substrate. The K_M and V_max values of the enzyme were 4.4 mg/ml and 287 U/mg respectively. At high xylan concentrations (>70 mg/ml) a substrate inhibition phenomenon of the enzyme was observed. In addition, crude xylanase showed enormous potential for decolorization of various recalcitrant dyes.     

Kinetic studies of the reduction of hexaaquacobalt(III) perchlorate by a cobaloxime, [Co(dmgBF2)2(H2O)2]

The reaction of with Co(dmgBF₂)₂(H₂O)₂ in 1.0 M HClO₄/LiClO₄ was found to be first-order in both reactants and the [H⁺] dependence of the second-order rate constant is given by k_2obs = b/[H⁺], b at 25 °C is 9.23 ± 0.14 × 10² s⁻¹. The [H⁺] dependence at lower temperatures shows some saturation effect that allowed an estimate of the hydrolysis constant for as K_a = 9.5 × 10⁻³ M at 10 and 15 °C. Marcus theory and the known self-exchange rate constant for Co(OH₂)₅OH^2+/+ were used to estimate an electron self-exchange rate constant of k₂₂ = 1.7 × 10⁻⁴ M⁻¹ s⁻¹ for .     

Longevity and temperature response of pollen as affected by elevated growth temperature and carbon dioxide in peanut and grain sorghum

It is important to understand the effects of environmental conditions during plant growth on longevity and temperature response of pollen. Objectives of this study were to determine the influence of growth temperature and/or carbon dioxide (CO₂) concentration on pollen longevity and temperature response of peanut and grain sorghum pollen. Plants were grown at daytime maximum/nighttime minimum temperatures of 32/22, 36/26, 40/30 and 44/34 °C at ambient (350 μmol mol⁻¹) and at elevated (700 μmol mol⁻¹) CO₂ from emergence to maturity. At flowering, pollen longevity was estimated by measuring in vitro pollen germination at different time intervals after anther dehiscence. Temperature response of pollen was measured by germinating pollen on artificial growth medium at temperatures ranging from 12 to 48 °C in incubators at 4 °C intervals. Elevated growth temperature decreased pollen germination percentage in both crop species. Sorghum pollen had shorter longevity than peanut pollen. There was no influence of CO₂ on pollen longevity. Pollen longevity of sorghum at 36/26 °C was about 2 h shorter than at 32/22 °C. There was no effect of growth temperature or CO₂ on cardinal temperatures (T_min, T_opt, and T_max) of pollen in both crop species. The T_min, T_opt, and T_max identified at different growth temperatures and CO₂ levels were similar at 14.9, 30.1, and 45.6 °C, respectively for peanut pollen. The corresponding values for sorghum pollen were 17.2, 29.4, and 41.7 °C. In conclusion, pollen longevity and pollen germination percentage was decreased by growth at elevated temperature, and pollen developed at elevated temperature and/or elevated CO₂ did not have greater temperature tolerance.     

Complex formation between protactinium(V) and sulfate ions at 10 and 60 °C

Protactinium complexation with sulfate ions was studied with the element at tracer scale (C_Pa ∼ 10⁻¹² M) by solvent extraction method. The involved aqueous system was Pa(V)/H₂O/HClO₄/Na₂SO₄/NaClO₄ at 10 and 60 °C. The extraction experiments were conducted using the chelating agent thenoyltrifluoroacetone (TTA) in toluene. For both values of temperature, a systematic study was performed in order to determine the formation constants (β₁, β₂ and β₃) of sulfate complexes of Pa(V) at different ionic strength. For each temperature, the extrapolation of these constants to zero ionic strength was performed using the Specific Interaction Theory, leading to values of 2.8 ± 0.5, 6.5 ± 0.5, 7.8 ± 0.5 at 10 °C and 4.3 ± 0.3, 8.4 ± 1.3, 9.6 ± 0.4 at 60 °C. Interaction coefficients involving the sulfate complexes of protactinium(V) were also derived.     

Chemical structures of water-soluble polysaccharides from Rhizoma Panacis Japonici

Five polysaccharide samples, coded as RPS1, RPS2, RPS3, RPS4, and RPS5, were isolated stepwise from Rhizoma Panacis Japonici (RPJ) by using 0.15 M NaCl aqueous solution at 25 °C, boiling water at 120 °C, 0.5 M NaOH/0.01 M NaBH₄ at 10 °C, 1.0 M NaOH/0.02 M NaBH₄ at 10 °C, and 19 M HCOOH at 4 °C, respectively. The yields were 0.39%, 1.08%, 2.41%, 0.32%, and 0.04% for RPS1 to RPS5, respectively. The chemical structures of the polysaccharides were highly branched α-(1→4)-d-glucan heteropolysaccharides and the values of degree of branch (DB) were in the range of 35-45% for RPS1 to RPS5. All of the polysaccharides were water soluble, and their solubility decreased from RPS1 to RPS5. The weight average molecular mass were 3.5 × 10⁴, 1.47 × 10⁵, 1.24 × 10⁶, 9.26 × 10⁵, and 1.36 × 10⁶ for RPS1 to RPS5, respectively.     

Heart valve cryopreservation: protocol for addition of dimethyl sulphoxide and amelioration of putative amphotericin B toxicity

Aim

To investigate the need for stepwise addition of dimethyl sulphoxide to heart valves and amelioration of putative amphotericin B toxicity.

Methods

There were four groups: an untreated control (Group 1) and three experimental groups. For the latter, porcine heart valves were exposed to the antibiotic/antimycotic mixture used for disinfecting heart valves in the Bristol Heart Valve Bank, for 24 h at 22 °C. Dimethyl sulphoxide (Me₂SO, 10% v/v) was added either in two steps (5% then 10%) (Group 2) or in a single step. For single-step addition, valves were either first placed in Hanks’ balanced salt solution for 10 min before transfer to the cryoprotectant solution (Group 3) or immersed directly in the 10% cryoprotectant solution (Group 4). The valve leaflets were dissected from the valves and frozen in 10% Me₂SO in multi-well tissue culture plates at 1 °C/min to −80 °C. After storage overnight, the valve leaflets were warmed at approximately 11 °C/min and the cryoprotectant was removed by single-step dilution in excess Hartmann’s solution. Each leaflet was then divided into four pieces, which were placed in separate wells of a culture plate. Outgrowth of cells from the explants was monitored daily and graded according to the extent of cell growth.

Results

After freezing and thawing, only 77% of the explants from valves placed directly into 10% Me₂SO (Group 4) showed outgrowth of cells after freezing compared with 89% with two-step addition of Me₂SO (Group 2) and 95% with one-step addition after the extra rinse in Hanks’ solution (Group 3) (χ², p = 0.001). 92% of unfrozen control explants showed outgrowth of cells (Group 1). Only 37% of Group 4 explants reached confluence compared with 63 and 56%, respectively, of Groups 2 and 3 explants (χ², p = 0.007). The rates of cell growth in Group 2 (two-step addition of Me₂SO) and Group 3 (one-step addition of Me₂SO with additional Hanks’ solution rinse) were similar and faster than the Group 4 (one-step addition of Me₂SO without the additional Hanks’ rinse).

Conclusion

Single-step addition of Me₂SO before freezing gave similar results to two-step addition provided an additional rinse in Hanks’ solution was introduced after exposure to the antibiotic/antimycotic mixture. This suggests that antibiotic/antimycotic carryover may have been harmful during freezing and that the additional rinse in Hanks before one-step addition of Me₂SO, and the 5% Me₂SO step in the two-step protocol, merely served to reduce this carryover.     

Seed germination of Lasia spinosa as a function of temperature,light, desiccation,and storage

Lasia spinosa seeds were not dormant at maturity in early spring. The most favorable temperatures for germination were between 25 and 30 °C, and final percentage and rate of germination decreased with an increase or decrease in temperature. When L. spinosa seeds were transferred to 25 °C, after 60 days at 10 °C (where none of the seeds germinated), final germination increased from 0% to 78%. Seeds germinated to high percentage both in light and in dark, although dark germination took more than twice as long as in the light. During desiccation of seeds at 15 °C and 45% relatively humidity, moisture loss decreased exponentially from 2.02 to 0.13 g H₂O g⁻¹ dry wt within 16 days, and only a few seeds (12%) survived 0.13 g H₂O g⁻¹ dry wt moisture content. Seeds stored at 0.58 g H₂O g⁻¹ dry wt moisture content at four constant temperatures (4, 10, 15, and −18 °C) for up to 6 months exhibited a well-defined trend of decreasing viability with decreasing temperature. Thus, we concluded that freshly harvested L. spinosa seeds are non-dormant and recalcitrant. Also, the seeds with 0.58 g H₂O g⁻¹ dry wt moisture content could be effectively stored for a few months between 10 and 15 °C although the most appropriate temperature for wet storage appears to be 10 °C, as it is close to the minimum temperature for germination and so there will be less pre-sprouting compared to 15 °C.     

Temperature manifold for a stopped-flow machine to allow measurements from −10 to +40 °C

Conducting enzymatic stopped-flow experiments at temperatures far removed from ambient can be very problematic because extremes in temperature (<10 °C or >30 °C) can damage the machine or the enzyme. We have devised a simple manifold that can be attached to most commercial stopped-flow systems that is independently heated or cooled separate from the main stopped-flow system. Careful calibration of the flow circuit allows the sample to be heated or cooled to the measurement temperature (−8 to +40 °C) 1 to 2 s before mixing in the reaction chamber. This approach allows measurements at temperatures where the stopped flow or the protein is normally unstable. To validate the manifold, we investigated the well-defined ATP-induced dissociation of rabbit muscle myosin subfragment 1 (S1) from its complex with pyrene-labeled actin. This process has both temperature-dependent and -independent components. Use of ethylene glycol allowed us to measure the reaction below 0 °C and up to 42 °C, and as expected the second-order rate constant (K₁k₊₂) and the maximum rate of dissociation (k₊₂) both increased with temperature, whereas 1/K₁ is unaffected by the change in temperature.     

Kinetics and mechanism of the reaction of octacarbonyl dicobalt with ethyl diazoacetate

Kinetics of the reaction of octacarbonyl dicobalt with ethyl diazoacetate leading to [μ²-{ethoxycarbonyl(methylene)}-μ²-(carbonyl)-bis(tricarbonyl-cobalt)] (Co-Co) (1), dinitrogen, and carbon monoxide were investigated at 10 °C in heptane solution. The initial rate of the reaction was measured by following both the gas evolution and the decrease of the octacarbonyl dicobalt concentration. The rate is first order with respect to octacarbonyl dicobalt and a complex order with respect to ethyl diazoacetate and carbon monoxide depending on the ratio of their concentrations. This is in accord with the formation of a heptacarbonyl dicobalt reactive intermediate (k₁ (10 °C) = (1.22 ± 0.06) × 10⁻³ s⁻¹) for which carbon monoxide and ethyl diazoacetate compete (k₋₁/k₂ (10 °C) = 1.34 ± 0.07).     

Characterization of lignin-rich residues remaining after continuous super-critical water hydrolysis of poplar wood (Populus albaglandulosa) for conversion to fermentable sugars

Poplar wood flour (Populous albaglandulosa) was treated with sub- and super-critical water (subcritical: 325, 350 °C; super-critical: 380, 400, 425 °C) for 60 s at 220 ± 10 atm. Hydrochloric acid (0.05% v/v) was added to samples as acidic catalyst. The final products were separated into water soluble fraction and undegraded solids. The yields of undegraded solids were thoroughly dependent on temperature severity and mainly composed of lignin fragments. Average molecular weights of the lignins were between 1500 and 4400 Da, which was only 1/3-1/8-fold of poplar milled wood lignin (13,250 Da). DFRC (Derivatization Followed by Reductive Cleavage) analysis revealed that C6C3 phenols (coniferyl and sinapyl alcohol) were rarely detected in the lignins, indicating occurrence of two probable lignin reactions during SCW hydrolysis: lignin fragmentation via splitting of β-O-4 linkage and loss of propane side chains. These results were also confirmed by ¹H and ¹³C NMR spectroscopic analysis.     

Intraspecific tolerance of Metarhizium anisopliae conidia to the upper thermal limits of summer with a description of a quantitative assay system

Conidial tolerance to the upper thermal limits of summer is important for fungal biocontrol agents, whose conidia are formulated into mycoinsecticides for field application. To develop an efficient assay system, aerial conidia of eight Metarhizium anisopliae, four M. anisopliae var. anisopliae, and six M. anisopliae var. acridum isolates with different host and geographic origins were wet-stressed for ≤180 min at 48 °C or incubated for 14 d colony growths at 10-35 °C. The survival ratios (relative to unstressed conidia) of each isolate, examined at 15-min intervals, fit a logistic equation (r² ≥ 0.975), yielding median lethal times (LT₅₀s) of 14.3-150.3 min for the 18 isolates stressed at 48 °C. Seven grasshopper isolates from Africa had a mean LT₅₀ of 110 (73-150) min, but could not grow at 10 or 15 °C. The mean LT₅₀ of five non-grasshopper isolates capable of growing at 10-35 °C was 16 (10-26) min only. Three isolates with typically low (type I), medium (type II), and high (type III) levels of tolerance to 48 °C were further assayed for ≤4-d tolerance of their conidia to the wet stress at 38, 40, 42, or 45 °C. The resultant LT₅₀s decreased to 20, 53 and 167 min at 48 °C from 507, 1612, and 8256 min at 38 °C for types I, II and III, respectively. For the distinguished types, the logarithms of the LT₅₀s were significantly correlated to the temperatures of 38-48 °C with an inverse linearity (r² ≥ 0.88). The method developed to assay quantitatively fungal thermotolerance would be useful for screening of fungal candidates for improved pest control in summer.     

Spermatozoa from the maned wolf (Chrysocyon brachyurus) display typical canid hyper-sensitivity to osmotic and freezing-induced injury,but respond favorably to dimethyl sulfoxide

We assessed the influences of medium osmolality, cryoprotectant and cooling and warming rate on maned wolf (Chrysocyon brachyurus) spermatozoa. Ejaculates were exposed to Ham’s F10 medium (isotonic control) or to this medium plus NaCl (350–1000 mOsm), sucrose (369 and 479 mOsm), 1 M glycerol (1086 mOsm) or dimethyl sulfoxide (Me₂SO, 1151 mOsm) for 10 min. Each sample then was diluted back into Ham’s medium and assessed for sperm motility and plasma membrane integrity. Although glycerol and Me₂SO had no influence (P > 0.05), NaCl and sucrose solutions affected sperm motility (P < 0.05), but not membrane integrity. Motility of sperm exposed to <600 mOsm NaCl or sucrose was less (P < 0.05) than fresh ejaculate, but comparable (P > 0.05) to the control. As osmolality of the NaCl solution increased, motility decreased to <5%. In a separate study, ejaculates were diluted in Test Yolk Buffer containing 1 M glycerol or Me₂SO and cooled from 5 °C to −120 °C at −57.8 °C, −124.2 °C or −67.0 °C/min, frozen in LN₂, thawed in a water bath for 30 s at 37 °C or 10 s at 50 °C, and then assessed for motility, plasma- and acrosomal membrane integrity. Cryopreservation markedly (P < 0.05) reduced sperm motility by 70% compared to fresh samples. Higher (P < 0.05) post-thaw motility (20.0 ± 1.9% versus 13.5 ± 2.1%) and membrane integrity (51.2 ± 1.7% versus 41.5 ± 2.2%) were observed in samples cryopreserved in Me₂SO than in glycerol. Cooling rates influenced survival of sperm cryopreserved in glycerol with −57.8 °C/min being advantageous (P < 0.05). The findings demonstrate that although maned wolf spermatozoa are similar to domestic dog sperm in their sensitivity to osmotic-induced motility damage, the plasma membranes tolerate dehydration, and the cells respond favorably to Me₂SO as a cryoprotectant.