Preparation of crystalline starch nanoparticles using cold acid hydrolysis and ultrasonication

Waxy maize starch in an aqueous sulfuric acid solution (3.16 M, 14.7% solids) was hydrolyzed for 2–6 days, either isothermally at 40 °C or 4 °C, or at cycled temperatures of 4 and 40 °C (1 day each). The starch hydrolyzates were recovered as precipitates after centrifuging the dispersion (10,000 rpm, 10 min). The yield of starch hydrolyzates depended on the hydrolysis temperature and time, which varied from 6.8% to 78%. The starch hydrolyzed at 40 °C or 4/40 °C exhibited increased crystallinity determined by X-ray diffraction analysis, but melted in broader temperature range (from 60 °C to 110 °C). However, the starch hydrolyzed at 4 °C displayed the crystallinity and melting endotherm similar to those of native starch. The starch hydrolyzates recovered by centrifugation were re-dispersed in water (15% solids), and the dispersion was treated by an ultrasonic treatment (60% amplitude, 3 min). The ultrasonication effectively fragmented the starch hydrolyzates to nanoparticles. The hydrolyzates obtained after 6 days of hydrolysis were more resistant to the ultrasonication than those after 2 or 4 days, regardless of hydrolysis temperatures. The starch nanoparticles could be prepared with high yield (78%) and crystallinity by 4 °C hydrolysis for 6 days followed by ultrasonication. Scanning electron microscopy revealed that the starch nanoparticles had globular shapes with diameters ranging from 50 to 90 nm.     

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.     

Simplified process for ethanol production from sugarcane bagasse using hydrolysate-resistant Escherichia coli strain MM160

Hexose and pentose sugars from phosphoric acid pretreated sugarcane bagasse were co-fermented to ethanol in a single vessel (SScF), eliminating process steps for solid-liquid separation and sugar cleanup. An initial liquefaction step (L) with cellulase was included to improve mixing and saccharification (L + SScF), analogous to a corn ethanol process. Fermentation was enabled by the development of a hydrolysate-resistant mutant of Escherichia coli LY180, designated MM160. Strain MM160 was more resistant than the parent to inhibitors (furfural, 5-hydroxymethylfurfural, and acetate) formed during pretreatment. Bagasse slurries containing 10% and 14% dry weight (fiber plus solubles) were tested using pretreatment temperatures of 160-190 °C (1% phosphoric acid, 10 min). Enzymatic saccharification and inhibitor production both increased with pretreatment temperature. The highest titer (30 g/L ethanol) and yield (0.21 g ethanol/g bagasse dry weight) were obtained after incubation for 122 h using 14% dry weight slurries of pretreated bagasse (180 °C).     

Kinetics and bioreactor studies of immobilized invertase on polyurethane rigid adhesive foam

A new support, polyurethane rigid adhesive foam (PRAF), which can be used to cover internal surface of metallic tubes, was used to immobilize invertase for application in an enzymatic bioreactor. The kinetic parameters were: K_m - 46.5 ± 1.9 mM (PRAF-invertase) and 61.2 ± 0.1 mM (free enzyme) and V_max 42.0 ± 4.3 U/mg protein/min (PRAF-invertase) and 445.3 ± 24.0 U/mg protein/min (free invertase). The PRAF-invertase derivative maintained 50.1% of initial activity (69.17 U/g support) for 8 months (4 °C) and was not observed microbial contamination. The bioreactor showed the best production of inverted sugar syrup using up-flow rate (0.48 L/h) with average conversion of 10.64 ± 1.5% h⁻¹ at feeding rate (D) of 104 h⁻¹. The operational inactivation rate constant (k_opi) and half-life were 1.92 × 10⁻⁴ min⁻¹ and 60 h (continue use). The PRAF spray support looks promising as a new alternative to produce immobilized derivatives on reactor surfaces.     

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.     

Injection of air into the headspace improves fermentation of phosphoric acid pretreated sugarcane bagasse by Escherichia coli MM170

Microaeration (injecting air into the headspace) improved the fermentation of hemicellulose hydrolysates obtained from the phosphoric acid pretreatment of sugarcane bagasse at 170 °C for 10 min. In addition, with 10% slurries of phosphoric acid pretreated bagasse (180 °C, 10 min), air injection into the headspace promoted xylose utilization and increased ethanol yields from 0.16 to 0.20 g ethanol/g bagasse dry weight using a liquefaction plus simultaneous saccharification and co-fermentation process (L+SScF). This process was scaled up to 80 L using slurries of acid pretreated bagasse (96 h incubation; 0.6 L of air/min into the headspace) with ethanol yields of 312-347 L (82-92 gal) per tonne (dry matter), corresponding to 0.25 and 0.27 g/g bagasse (dry weight). Injection of small amounts of air into the headspace may provide a convenient alternative to subsurface sparging that avoids problems of foaming, sparger hygiene, flotation of particulates, and phase separation.     

Supercritical carbon dioxide pretreatment of corn stover and switchgrass for lignocellulosic ethanol production

Supercritical CO₂ (SC-CO₂), a green solvent suitable for a mobile lignocellulosic biomass processor, was used to pretreat corn stover and switchgrass at various temperatures and pressures. The CO₂ pressure was released as quickly as possible by opening a quick release valve during the pretreatment. The biomass was hydrolyzed after pretreatment using cellulase combined with β-glucosidase. The hydrolysate was analyzed for the amount of glucose released. Glucose yields from corn stover samples pretreated with SC-CO₂ were higher than the untreated sample’s 12% glucose yield (12 g/100 g dry biomass) and the highest glucose yield of 30% was achieved with SC-CO₂ pretreatment at 3500 psi and 150 °C for 60 min. The pretreatment method showed very limited improvement (14% vs. 12%) in glucose yield for switchgrass. X-ray diffraction results indicated no change in crystallinity of the SC-CO₂ treated corn stover when compared to the untreated, while SEM images showed an increase in surface area.     

Purification and enzymatic characterization of two β-endoxylanases from Trichoderma sp. K9301 and their actions in xylooligosaccharide production

Trichoderma sp. K9301 secreting endoxylanases with an activity of 2836 U/g (dry weight) was screened for XOs production. Two acidic β-endoxylanases EX1 (30.1 kDa) and EX2 (20.1 kDa) were purified from crude extract of the strain K9301 in solid fermentation. Action modes of EX1 and EX2 towards XOs showed similar hydrolysis characters to endoxylanases belonging to glycosyl hydrolase family 10 and 11, respectively. EX1 exhibited better affinity but lower hydrolytic efficiency than EX2 to xylans from beechwood, birchwood, and oat-spelt. They had synergistic action on xylan hydrolysis. The optimum condition to prepare XOs from corncobs was obtained as 10 mg/ml corncob xylan incubated with 10 U/mg crude enzymes at 50 °C for 3 h. The yield of XOs reached 43.3%, and only a little amount of xylose (3.1%) was simultaneously produced, suggesting the good potential of strain K9301 in XOs production.     

Effect of operating conditions of thermochemical liquefaction on biocrude production from Spirulina platensis

This study investigated the optimum thermochemical liquefaction (TCL) operating conditions for producing biocrude from Spirulina platensis. TCL experiments were performed at various temperatures (200-380 °C), holding times (0-120 min), and solids concentrations (10-50%). TCL conversion at 350 °C, 60 min holding time and 20% solids concentration produced the highest biocrude yield of 39.9% representing 98.3% carbon conversion efficiency. Light fraction biocrude (B₁) appeared at 300 °C or higher temperatures and represented 50-63% of the total biocrude. Biocrude obtained at 350-380 °C had similar fuel properties to that of petroleum crude with energy density of 34.7-39.9 MJ kg⁻¹ compared to 42.9 MJ kg⁻¹ for petroleum crude. Biocrude from conversion at 300 °C or above had 71-77% elemental carbon, and 0.6-11.6% elemental oxygen and viscosities in the range 40-68 cP. GC/MS of biocrude reported higher hydrocarbons (C₁₆-C₁₇), phenolics, carboxylic acids, esters, aldehydes, amines, and amides.     

Definition and characterization of enzymes for maximal biocatalytic solubilization of prebiotic polysaccharides from potato pulp

Potato pulp is a high-volume co-processing product resulting from industrial potato starch manufacturing. Potato pulp is particularly rich in pectin, notably galactan branched rhamnogalacturonan I polysaccharides, which are highly bifidogenic when solubilized. The objective of the present study was to characterize and compare four homogalacturonan degrading enzymes capable of catalyzing the required solubilization of these pectinaceous polysaccharides from potato pulp in a 1 min reaction. An additional purpose was to assess the influence of the pH and the potential buffer chelating effects on the release of these polysaccharides from the potato pulp. The pH and temperature optima of two selected pectin lyases from Emericella nidulans (formerly known as Aspergillus nidulans) and Aspergillus niger were determined to 8.6 and 4.0, respectively, at ≥100 °C within 1 min of reaction. The optima for the two selected polygalacturonases from E. nidulans and Aspergillus aculeatus were determined to pH 4.4 and 46 °C, and pH 3.7 and ≥80 °C, respectively. The polygalacturonase from A. aculeatus was 4-42 times more heat-resistant at 50 °C than the other enzymes. The difference in pH optima of the pectin lyases and the exceptional thermal stabilities of some of the enzymes are proposed to be related to specific amino acid substitutions, stabilizing hydrogen bonding and structural traits of the enzymes. The K_M and V_max values ranged from 0.3-0.6 g/L and 0.5-250.5 U/mg protein, respectively. Phosphate buffer induced release of a higher amount of dry matter than Tris-acetate buffer at pH 6, indicating a chelating effect of the phosphate. Moreover, the phosphate had a higher chelating effect at pH 6 than at pH 4. The optimal conditions for a high yield of polysaccharides from potato pulp were therefore: 1% (w/w) potato pulp treated with 1% (w/w) enzyme/substrate (E/S) pectin lyase from E. nidulans and 1% (w/w) E/S polygalacturonase from A. aculeatus at pH 6.0 and 60 °C for 1 min.     

Production of furans from rice straw by single-phase and biphasic systems

5-Hydroxymethylfurfural (HMF) and furfural, both of which can be derived from renewable sources, are key components for the production of different chemicals and fuels. In this study, rice straw, a cheap, abundant, and mainly unused agricultural waste, is converted to furans by a dilute acid hydrolysis process. The highest yield of HMF in a single-phase hydrolysis was 15.3 g/kg straw, attained at 180 °C during 3 h with 0.5% sulfuric acid, while the maximum yield of furfural, 59 g/kg straw, was obtained at 150 °C during 5 h. Different extracting solvents, including 2-PrOH, 1-BuOH, methyl isobutyl ketone (MIBK), and acetone at 180 °C for 3 h as well as tetrahydrofuran (THF) at 150 °C for 5 h were examined in biphasic systems. Use of the solvents generally improved the production of HMF compared to the single aqueous phase process. The best results of HMF production, more than 59 g/kg straw, were obtained in the systems containing either 2-PrOH or 1-BuOH. Using THF as an extracting solvent, a relatively high furfural yield, 118.2 g/kg straw, was obtained, and 96% of furfural produced in this system was extracted into THF during the process.     

Liquid hot water and alkaline pretreatment of soybean straw for improving cellulose digestibility

Soybean straw was pretreated with either liquid hot water (LHW) (170-210 °C for 3-10 min) or alkaline soaking (4-40 g NaOH/100 g dry straw) at room temperature to evaluate the effects on cellulose digestibility. Nearly 100% cellulose was recovered in pretreated solids for both pretreatment methods. For LHW pretreatment, xylan dissolution from the raw material increased with pretreatment temperature and time. Cellulose digestibility was correlated with xylan dissolution. A maximal glucose yield of 70.76%, corresponding to 80% xylan removal, was obtained with soybean straw pretreated at 210 °C for 10 min. NaOH soaking at ambient conditions removed xylan up to 46.37% and the subsequent glucose yield of pretreated solids reached up to 64.55%. Our results indicated LHW pretreatment was more effective than NaOH soaking for improving cellulose digestibility of soybean straw.     

Pretreatment and enzymic saccharification of water hyacinth cellulose

Water hyacinth was pretreated, under variable conditions, with NaOH, alkaline H₂O₂, peracetic acid and sodium chlorite. Combined pretreatments included sodium chlorite with each of NaOH, alkaline H₂O₂ and peracetic acid. Combined pretreatment with 0.1% NaClO₂ for 1 h at 100 °C and peracetic acid at 100 °C for 15 min afforded the most promising sample. The recovered lignin, cellulose and hemicellulose of this sample was 2.56%, 96.69%, and 81.38%, respectively. The same sample, by cellulase hydrolysis showed the highest cellulose conversion (80.8%) and 90% saccharification using 200 FPU/g substrate. Some ambient factors affecting saccharification of pretreated water hyacinth were investigated. Enzymic saccharification after 6 h was about 50% of that at 48 h, indicating a slow hydrolysis rate by time. Addition of 8% glucose at the beginning of the enzymic hydrolysis decreased the saccharification to about its half while addition of 8% ethanol brought about complete inhibition of the enzyme. Addition of cellobiase to the reaction mixture increased cellulose conversion and saccharification by 10%.     

Optimizing pentose utilization in yeast: the need for novel tools and approaches

Background

The two-step dilute acid hydrolysis (DAH) of softwood is costly in energy demands and capital costs. However, it has the advantage that hydrolysis and subsequent removal of hemicellulose-derived sugars can be carried out under conditions of low severity, resulting in a reduction in the level of sugar degradation products during the more severe subsequent steps of cellulose hydrolysis. In this paper, we discuss a single-step DAH method that incorporates a temperature profile at two levels. This profile should simulate the two-step process while removing its major disadvantage, that is, the washing step between the runs, which leads to increased energy demand.

Results

The experiments were conducted in a reactor with a controlled temperature profile. The total dry matter content of the hydrolysate was up to 21.1% w/w, corresponding to a content of 15.5% w/w of water insoluble solids. The highest measured glucose yield, (18.3 g glucose per 100 g dry raw material), was obtained after DAH cycles of 3 min at 209°C and 6 min at 211°C with 1% H₂SO₄, which resulted in a total of 26.3 g solubilized C6 sugars per 100 g dry raw material. To estimate the remaining sugar potential, enzymatic hydrolysis (EH) of the solid fraction was also performed. EH of the solid residue increased the total level of solubilized C6 sugars to a maximum of 35.5 g per 100 g dry raw material when DAH was performed as described above (3 min at 210°C and 2 min at 211°C with 1% H₂SO₄).

Conclusion

The dual-temperature DAH method did not yield decisively better results than the single-temperature, one-step DAH. When we compared the results with those of earlier studies, the hydrolysis performance was better than with the one-step DAH but not as well as that of the two-step, single-temperature DAH. Additional enzymatic hydrolysis resulted in lower levels of solubilized sugars compared with other studies on one-step DAH and two-step DAH followed by enzymatic hydrolysis. A two-step steam pretreatment with EH gave rise to a considerably higher sugar yield in this study.     

Cellulose isolation and core-shell nanostructures of cellulose nanocrystals from chardonnay grape skins

This is the first report on the derivation and structures of cellulose nanocrystals from grape skins. Pure cellulose was isolated from chardonnay grape skins at a 16.4% yield by a process involving organic extraction, acid and base dissolutions, and basic and acidic oxidation. The as-extracted cellulose was 54.9% crystalline and microfibrillar. Acid hydrolysis (64-65% H₂SO₄ 45 °C, 30 min) of grape skin cellulose produced the more crystalline (64.3%) cellulose nanocrystals (CNCs) that appeared mostly as spherical nanoparticles with diameters ranging from 10 to 100 nm and a mean diameter of 48.1 (±14.6) nm as observed by TEM. AFM further disclosed the spherical nanoparticles actually consist of a nano-rod core (seed) surrounded by numerous tiny cellulose fragments as the shell. Interestingly, the spherical core-shell nanoparticles resemble the shape of grape bundles, the starting biomass, may be assembled via interfacial hydrogen bonds.     

Shivering modulation in humans: Effects of rapid changes in environmental temperature

During cold exposure, increase in heat production is produced via the activation of shivering thermogenesis and nonshivering thermogenesis, the former being the main contributor to compensatory heat production in non-acclimatized humans. In rats, it has been demonstrated that shivering thermogenesis is modulated solely by skin thermoreceptors but this modulation has yet to be investigated in humans. The aim of this study was to determine if cold-induced shivering in humans can be modulated by cutaneous thermoreceptors in conditions where increases in heat loss can be adequately compensated by increases in thermogenic rate. Using a liquid-conditioned suit, six non-acclimatized men were exposed to cold (6 °C) for four 30 min periods, each of them separated by 15 min of heat exposure (33 °C). Core temperature remained stable throughout exposures whereas skin temperatures significantly decreased by 12% in average during the sequential cold/heat exposures compared to baseline (p<0.0001). Shivering intensity and metabolic rate increased significantly during 6 °C exposures (3.3±0.7% MVC, 0.40±0.0 L O₂/min, respectively) and were significantly reduced during 33 °C exposure (0.5±0.1% MVC, 0.25±0.0 L O₂/min; p<0.005 for both). Most importantly, shivering could be quickly and strongly inhibited during 33 °C exposure although skin temperature often remained below baseline values. In conclusion, under compensatory conditions, cutaneous thermoreceptors appear to be a major modulator of the shivering response in humans and seem to react rapidly to changes in the microclimate right next to the skin and to skin temperature.     

One-step production of biodiesel from Jatropha oil with high-acid value in ionic liquids

Catalytic conversion of un-pretreated Jatropha oil with high-acid value (13.8 mg KOH/g) to biodiesel was studied in ionic liquids (ILs) with metal chlorides. Several commercial ILs were used to catalyze the esterification of oleic acid. It was found that 1-butyl-3-methylimidazolium tosylate ([BMIm][CH₃SO₃]; a Brønsted acidic IL) had the highest catalytic activity with 93% esterification rate for oleic acid at 140 °C but only 12% biodiesel yield at 120 °C. When FeCl₃ was added to [BMIm][CH₃SO₃], a maximum biodiesel yield of 99.7% was achieved at 120 °C. Because metal ions in ILs supplied Lewis acidic sites, and more of the sites could be provided by trivalent metallic ions than those of bivalent ones. It was also found that the catalytic activity with bivalent metallic ions increased with atomic radius. Mixture of [BMIm][CH₃SO₃] and FeCl₃ was easily separated from products for reuse to avoid producing pollutants.     

Control of choline oxidation in rat kidney mitochondria

Choline is a quaternary amino cationic organic alcohol that is oxidized to betaine in liver and kidney mitochondria. Betaine acts as an intracellular organic osmolyte in the medulla of the kidney. Evidence is provided that kidney mitochondria have a choline transporter in their inner membrane. The transporter has a Km of 173 ± 64 μM and a Vmax of 0.4 ± 0.1 nmol/min/mg mitochondrial protein (at 10 °C). Uptake of choline is not coupled to betaine efflux. Transporter activity demonstrates a dependence on membrane potential and choline transport is inhibited by hemicholinium-3. Steady-state oxygen consumption due to choline oxidation in kidney mitochondria was measurable at 37 °C (125 ± 6 pmolO₂/min/mg mitochondrial protein), in the absence of other mitochondrial electron transport chain substrates and the choline transporter was shown to be the major site of control (96 ± 4%) over choline oxidation flux in isolated kidney mitochondria. We conclude that the choline transporter in rat kidney mitochondria is the major site of control over the production of the organic osmolyte, betaine.     

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.     

Cryopreservation of red snapper (Lutjanus argentimaculatus) sperm: Effect of cryoprotectants and cooling rates on sperm motility, sperm viability, and fertilization capacity

Sperm cryopreservation of red snapper (Lutjanus argentimaculatus) is essentially unexplored, although many species of the Lutjanidae family are considered to be high-value commercial species. The objective of this study was to develop a species-specific cryopreservation protocol for red snapper (L. argentimaculatus) sperm by optimizing cryoprotectants and cooling rates in the cryopreservation procedure. Ten cryoprotectants at four concentrations and two freezing protocols were examined in two separate experiments. In the first experiment, toxicity studies of dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), ethylene glycol (EG), formamide, methanol, ethanol, sucrose, trehalose, and dimethylacetamide (DMA) on sperm motility were performed. Semen diluted 1:1 in Ringer solution were exposed to cryoprotectants at four final concentrations of 5%, 10%, 15%, or 20% for periods of 10, 20, 30, 40, 50, 60, 90, and 120 min at room temperature (25 °C). The cryoprotectants and concentrations that showed the least toxic effect on sperm motility were selected for cryopreservation trials. In the second experiment, selected cryoprotectants were then assessed for freezing capacity of sperm as follows: DMSO 5% and 10%, PG 5% and 10%, EG 5% and 10%, ethanol 5%, and methanol 5%. Semen was diluted 1:1 in Ringer solution and equilibrated with selected cryoprotectants for 10 min at room temperature. Sperm were frozen in a controlled-rate programmable freezer at four cooling rates of 3, 5, 10, and 12 °C/min from an initial temperature of 25 °C to final temperatures of −40 or −80 °C before plunging into liquid nitrogen. Sperm equilibrated in 10% DMSO and cooled at a rate of 10 °C/min to a final temperature of −80 °C had the highest motility (91.1 ± 2.2%) and viability (92.7 ± 2.3%) after thawing. The fertilization rate of frozen-thawed sperm (72.4 ± 2.4%) was not different (P > 0.05) from that of fresh sperm (75.5 ± 2.4%). This study apparently represents the first reported attempt for cryopreservation of L. argentimaculatus sperm.