Development of a modified vitrification strategy suitable for subsequent scale-up for hepatocyte preservation

This work explores the design of a vitrification solution (VS) for scaled-up cryopreservation of hepatocytes, by adapting VS_basic (40% (v/v) ethylene glycol 0.6 M sucrose, i.e. 7.17 M ethylene glycol 0.6 M sucrose), previously proven effective in vitrifying bioengineered constructs and stem cells. The initial section of the scale-up study involved the selection of non-penetrating additives to supplement VS_basic and increase the solution’s total solute concentration. This involved a systematic approach with a step-by-step elimination of non-penetrating cryoprotectants, based on their effect on cells after long/short term exposures to high/low concentrations of the additives alone or in combinations, on the attachment ability of hepatocytes after exposure. At a second stage, hepatocyte suspension was vitrified and functions were assessed after continuous culture up to 5 days.Results indicated Ficoll as the least toxic additive. Within 60 min, the exposure of hepatocytes to a solution composed of 9% Ficoll + 0.6 M sucrose (10⁻³ M Ficoll + 0.6 M sucrose) sustained attachment efficiency of 95%, similar to control. Furthermore, this additive did not cause any detriment to the attachment of these cells when supplementing the base vitrification solution VS_basic. The addition of 9% Ficoll, raised the total solute concentration to 74.06% (w/v) with a negligible 10⁻³ M increase in molarity of the solution. This suggests main factor in inducing detriment to cells was the molar contribution of the additive.Vitrification protocol for scale-up condition sustained hepatocyte suspension attachment efficiency and albumin production. We conclude that although established approach will permit scaling-up of vitrification of hepatocyte suspension, vitrification of hepatocytes which are attached prior to vitrification is more effective by comparison.     

Vitrification as a method for genome resource banking oocytes from the endangered Tasmanian devil (Sarcophilus harrisii)

Populations of Australia’s largest terrestrial marsupial carnivore, the Tasmanian devil (Sarcophilus harrisii), are rapidly declining in the wild due to Tasmanian Devil Facial Tumour Disease (TDFTD). One tool which can reduce the loss of genetic diversity is genome resource banking. This study examines the application of an oocyte vitrification protocol, initially developed in a model marsupial carnivore, to the endangered Tasmanian devil. Ovarian tissue was transported to the laboratory on ice from Tasmania which took up to 48 h. Individual granulosa oocyte complexes (GOC) were isolated enzymatically and the viability of oocytes from primary GOC was assessed immediately following isolation or after exposure to cold shock, vitrification and thawing media without exposure to liquid nitrogen or the full vitrification and thawing process. There was no decline in oocyte viability following cold shock or exposure to the vitrification and thawing media. Following the full vitrification and thawing process there was a decline in oocyte viability (χ² = 20.0, P < 0.001) but approximately 70% of oocytes remained viable. This study provides further evidence that oocyte vitrification is a promising strategy for genome resource banking in carnivorous marsupials and suggests that it should be considered in conservation plans for the survival of the iconic Tasmanian devil.     

Production of channel catfish with sperm cryopreserved by rapid non-equilibrium cooling

This report describes the feasibility of using vitrification for fish sperm. Vitrification can be used to preserve samples in the field and offers an alternative to conventional cryopreservation, although it has not been systematically studied for sperm of aquatic species. The overall goal of the project was to develop streamlined protocols that could be integrated into a standardized approach for vitrification of aquatic species germplasm. The objectives of the present study in channel catfish (Ictalurus punctatus) were to: (1) evaluate the acute toxicity of 5%, 10%, 20% and 30% methanol, N,N-dimethyl acetamide, dimethyl sulfoxide, 1,2-propanediol, and methyl glycol; (2) evaluate a range of devices commonly used for cryopreservation and vitrification of mammalian sperm; (3) compare vitrification with and without cryoprotectants; (4) evaluate the post-thaw membrane integrity of sperm vitrified in different cryoprotectant solutions, and (5) evaluate the ability of vitrified sperm to fertilize eggs. Cryoprotectant concentrations of higher than 20% were found to be toxic to sperm. Methanol and methyl glycol were the least toxic at a concentration of 20% with an exposure time of less than 5 min. We evaluated a method reported for human sperm, using small volumes in loops (15 μl) or cut standard straws (20 μl) with and without cryoprotectants plunged into liquid nitrogen. Cryoprotectant-free vitrification using loops did not yield fertilization (assessed by neurulation), and the fertilization rates observed in two trials using the cut standard straws were low (∼2%). In general, fertilization values for vitrification experiments were low and the use of low concentrations of cryoprotectants yielded lower fertilization (<10%) than the use of vitrification solutions containing high cryoprotectant concentrations (as high as 25%). The highest neurulation obtained was from a mixture of three cryoprotectants (20% methanol + 10% methyl glycol + 10% propanediol) with a single-step addition. This was reflected in the flow cytometry data from which the highest membrane integrity using loops was for 20% methanol + 10% methyl glycol + 10% propanediol (∼50%). We report the first successful sperm vitrification in fish and production of offspring from vitrified sperm in channel catfish. Although the fertilization values were low, at present this technique could nevertheless be used to reconstitute lines (especially in small aquarium fishes), but it would require improvement and scaling up before being useful as a production method for large-bodied fishes such as catfish.     

Relative larvicidal potentiality of nano-encapsulated Temephos and Imidacloprid against Culex quinquefasciatus

Encapsulation of temephos ranging from 1% to 16% and imidacloprid from 1% to 8% within biodegradable and biocompatible, polyethylene glycol in different ratios was done by using melt-dispersion method. The efficacy of encapsulated forms was evaluated and compared with their non-capsulated forms against larvae of Culex quinquefasciatus. The encapsulated temephos was more toxic than the encapsulated imidacloprid with LC₅₀ values of 0.013, 0.010 and 0.003 mg/L after 24, 48 and 72 h, respectively. No doubt, the non-capsulated temephos and imidacloprid were more effective as compared to their encapsulated forms. However, the same mortality rate was achieved by the slow release of lesser amount of pesticides after encapsulation, e.g., 0.003 mg/L for 8% temephos formulation and 0.019 mg/L for 4% imidacloprid as compared to their non-capsulated form of temephos and imidacloprid (0.004 and 0.021 mL/L) after 72 h of exposure. Thus, encapsulated forms are more economical and eco-friendly due to controlled slow release of their nanoparticles.     

Cryopreservation of human failed maturation oocytes shows that vitrification gives superior outcomes to slow cooling

This study investigated whether failed maturation oocytes could be used to evaluate different cryopreservation procedures. A total of 289 failed maturation oocytes (GV and MI stages), obtained from 169 patients undergoing IVF treatment (mean age 33.84 ± 5.0) were divided into two different slow-cooling groups (1.5 mol/l 1,2-propanediol + 0.2 mol/l sucrose in either NaCl (group A) or choline chloride (ChCl) (group B) based cryopreservation solutions) and one vitrification group (15% ethylene glycol + 15% dimethyl sulphoxide). Survival rate, in vitro maturation (IVM) rate, fertilization and developmental rate of cryopreserved oocytes were assessed. Regardless of the stage at which cryopreservation was performed (GV + MI), the slow cooling with ChCl based medium always gave significantly lower survival rate than the slow cooling in NaCl based medium (p = 0.01) and vitrification (p < 0.001). An extended study also showed statistically reduced survival rate between slow-cooling NaCl based medium and vitrification (p < 0.05). Global results of in vitro maturation and fertilization showed worse results between both slow-cooling NaCl and ChCl based media versus vitrification. In conclusion, for oocytes that had failed to mature, vitrification gave better survival, maturation, fertilization and also cleavage rates than the slow-cooling protocols. Four cells embryos were obtained only from vitrified in vitro matured MI oocytes.     

Cryopreservation of human failed maturation oocytes shows that vitrification gives superior outcomes to slow cooling

This study investigated whether failed maturation oocytes could be used to evaluate different cryopreservation procedures. A total of 289 failed maturation oocytes (GV and MI stages), obtained from 169 patients undergoing IVF treatment (mean age 33.84 ± 5.0) were divided into two different slow-cooling groups (1.5 mol/l 1,2-propanediol + 0.2 mol/l sucrose in either NaCl (group A) or choline chloride (ChCl) (group B) based cryopreservation solutions) and one vitrification group (15% ethylene glycol + 15% dimethyl sulphoxide). Survival rate, in vitro maturation (IVM) rate, fertilization and developmental rate of cryopreserved oocytes were assessed. Regardless of the stage at which cryopreservation was performed (GV + MI), the slow cooling with ChCl based medium always gave significantly lower survival rate than the slow cooling in NaCl based medium (p = 0.01) and vitrification (p < 0.001). An extended study also showed statistically reduced survival rate between slow-cooling NaCl based medium and vitrification (p < 0.05). Global results of in vitro maturation and fertilization showed worse results between both slow-cooling NaCl and ChCl based media versus vitrification. In conclusion, for oocytes that had failed to mature, vitrification gave better survival, maturation, fertilization and also cleavage rates than the slow-cooling protocols. Four cells embryos were obtained only from vitrified in vitro matured MI oocytes.     

Effect of the exposure to methyl-β-cyclodextrin prior to chilling or vitrification on the viability of bovine immature oocytes

The present study aimed to evaluate the effect of methyl-β-cyclodextrin (MβCD) as a cholesterol loader to change oocyte plasma membrane and increase its tolerance toward cryopreservation. The first and second experiments were conducted to investigate if MβCD could improve nuclear and cytoplasmic maturation after oocyte exposure to cold stress for 10 or 30 min, respectively. No differences (P > 0.05) in either experiment in the metaphase II (MII) rate of oocytes exposed to MβCD and cold stress; but these oocytes presented lower maturation rates than control groups. In the second experiment, a lower percentage of oocytes showed degenerated chromatin (P < 0.05) after exposure to 2 mg/mL of MβCD compared to the group exposed to 0 mg/mL. However, no differences among treatments were observed in cytoplasmic maturation. Groups exposed to cold stress demonstrated a lower (P < 0.05) capacity for embryonic development compared to the control groups. In the third experiment immature oocytes were exposed to MβCD and then, vitrified (cryotop). After warming, we observed that the ability to reach MII and chromatin degeneration were altered (P < 0.05) by MβCD. The blastocysts rate (P < 0.05) on D7 was higher in the 2 mg/mL MβCD group, but an identical finding was not observed on D8 (P > 0.05). Chromatin degeneration was higher in the vitrification groups. We conclude that MβCD improved nuclear maturation by reducing oocyte degeneration after cold stress or vitrification; however, more studies are required to clarify the usefulness of MβCD use in oocyte cryopreservation.     

The effect of encapsulated glutamine on gut peptide secretion in human volunteers

ContextWeight loss and improved blood glucose control after bariatric surgery have been attributed in part to increased ileal nutrient delivery with enhanced release of glucagon-like peptide 1 (GLP-1). Non-surgical strategies to manage obesity are required. The aim of the current study was to assess whether encapsulated glutamine, targeted to the ileum, could increase GLP-1 secretion, improve glucose tolerance or reduce meal size.MethodsA single-center, randomised, double blind, placebo-controlled, cross-over study was performed in 24 healthy volunteers and 8 patients with type 2 diabetes. Fasting participants received a single dose of encapsulated ileal-release glutamine (3.6 or 6.0 g) or placebo per visit with blood sampling at baseline and for 4 h thereafter. Glucose tolerance and meal size were studied using a 75 g oral glucose tolerance test and ad libitum meal respectively.ResultsIn healthy volunteers, ingestion of 6.0 g glutamine was associated with increased GLP-1 concentrations after 90 min compared with placebo (mean 10.6 pg/ml vs 6.9 pg/ml, p = 0.004), increased insulin concentrations after 90 min (mean 70.9 vs 48.5, p = 0.048), and increased meal size at 120 min (mean 542 g eaten vs 481 g, p = 0.008). Ingestion of 6.0 g glutamine was not associated with significant differences in GLP-1, glucose or insulin concentrations after a glucose tolerance test in healthy or type 2 diabetic participants.ConclusionsSingle oral dosing of encapsulated glutamine did not provoke consistent increases in GLP-1 and insulin secretion and was not associated with beneficial metabolic effects in healthy volunteers or patients with type 2 diabetes.     

Survival of tissue balls from the coral Pocillopora damicornis L. exposed to cryoprotectant solutions

In this study, the tolerance of tissue balls (TBs, 100–300 μm in diameter) from the coral Pocillopora damicornis produced using mechanical excision to exposure to cryoprotectant (CPA) solutions was tested. TBs were treated for 20 min at room temperature with solutions of ethylene glycol (EG), methanol (Met), glycerol (Gly) or dimethyl sulfoxide (Me₂SO) at concentrations between 1.0 and 4.5 M. Two parameters were used to evaluate the survival of TBs following CPA treatment. The Undamaged Duration of Tissue Balls (expressed in h) corresponded to the time period during which the membrane surface of TBs remained smooth and their motility was preserved. Tissue Ball Regression (expressed in μm/h) corresponded to the size reduction of TBs over time. TBs tolerated exposure to all CPAs tested at the three lower concentrations employed (1.0 M, 1.5 M and 2.0 M). No survival was achieved following exposure to a 4.5 M CPA solution. At concentrations of 3.0 and 4.0 M, higher Undamaged Duration of Tissue Balls and lower Tissue Ball Regression were obtained following treatment with EG compared to the other three CPAs. Our experiments show that TBs constitute a good experimental material to evaluate CPA toxicity on corals using large numbers of samples. Performing preliminary experiments with TBs may allow reducing the number of tests carried out with less easily available coral forms such as planulae, thereby preserving larval stocks.     

Developmental competence and gene expression of immature oocytes following liquid helium vitrification in bovine

The objective of this study was to develop an effective ultra-rapid vitrification method and evaluate its effect on maturation, developmental competence and development-related gene expression in bovine immature oocytes. Bovine cumulus oocyte complexes were randomly allocated into three groups: (1) controls, (2) liquid nitrogen vitrification, and (3) liquid helium vitrification. Oocytes were vitrified and then warmed, the percentage of morphologically normal oocytes in liquid helium group (89.0%) was significantly higher (P < 0.05) than that of the liquid nitrogen group (81.1%). When the vitrified–thawed oocytes were matured in vitro for 24 h, the maturation rate in liquid helium group (50.6%) was higher (P < 0.05) than liquid nitrogen group (42.6%). Oocytes of liquid helium vitrification had higher cleavage and blastocyst rates (41.1% and 10.0%) than that of liquid nitrogen vitrification (33.0% and 4.5%; P < 0.05) after in vitro fertilization. Moreover, the expression of GDF9 (growth/differentiation factor-9), BAX (apoptosis factor) and ZAR1 (zygote arrest 1) was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) when the vitrified–thawed oocytes were matured 24 h. The expression of these genes was altered after vitrification. Expression of GDF9 and BAX in the liquid helium vitrification group was not significantly different from that of the control, however there were significant differences between the liquid nitrogen vitrification group and control. In conclusion, it was feasible to use liquid helium for vitrifying bovine immature oocytes. There existed an association between the compromised developmental competence and the altered expression levels of these genes for the vitrified oocytes.     

Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity

Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxy-poly(ethylene glycol)-hydrazide (4.5 kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100 kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24 h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24 h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220 ± 73% signal change (n = 4, 95% confidence interval) over the physiologically-relevant glucose range (i.e., 0–400 mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay.     

Comparison of Ephestia kuehniella eggs sterilization methods for Trichogramma rearing

Mass production is necessary to ensure the availability of biological control agents for the suppression of target pests. Many rearing hosts need to be sterilized to prevent development. Host egg sterilization also allows their storage for a longer period. Ephestia kuehniella eggs are frequently used as hosts for Trichogramma parasitoïds but they must be sterilized to prevent larvae from emerging and eating the unhatched parasitized eggs. Three sterilization methods were examined: UV irradiation, freezing at −15 °C and vitrification (liquid nitrogen submersion). The dosage and exposure duration to provide egg sterilization were determined and then the suitability of hosts sterilized by the different methods were compared. E. kuehniella eggs abortion was achieved after 15 min by UV irradiation, 4 h by freezing at −15 °C and 30 s by vitrification. Vitrification resulted in significantly lower parasitoids production with a global emergence rate of 28.7%, compared to UV irradiation (75.1%), freezing at −15 °C (77.4%) and control (80.9%). Host eggs sterilization method did not affect sex-ratio, occurrence of malformation in adults, and female walking speed. Fecundity was significantly reduced in the females emerging from UV irradiated (37.2 offsprings) and vitrified (36.9 offsprings) eggs, compared to control (43.1 offsprings).     

Cold tolerance of the maize orange leafhopper,Cicadulina bipunctata

Cicadulina bipunctata was originally distributed in tropical and subtropical regions of the Old World. This leafhopper recently expanded its distribution area to southern parts of temperate Japan. In this study, factors affecting the overwintering ability of C. bipunctata were examined. A series of laboratory experiments revealed that cold acclimation at 15 °C for 7 days enhanced the cold tolerance of C. bipunctata to the same level as an overwintering population, adult females were more tolerant of cold temperature than adult males, and survival of acclimated adult females was highly dependent on temperature from −5 to 5 °C and exposure duration to the temperature. The temperature of crystallization of adult females was approximately −19 °C but temperatures in southern temperate Japan rarely dropped below −10 °C in the winter, indicating that overwintering C. bipunctata adults in temperate Japan are not killed by freezing injury but by indirect chilling injury caused by long-term exposure to moderately low temperatures. An overwintering generation of C. bipunctata had extremely low overwinter survival (<1%) in temperate Japan; however, based on winter temperature ranges, there are additional areas amenable to expansion of C. bipunctata in temperate Japan.     

Comparison between ambulatory measurement of effective thermal conductivity and laser Doppler flowmetry method to assess skin microcirculatory activity

The main objective of this paper was to assess the performance of the ambulatory device μHematron to measure indirectly skin blood flow relative to the well-established Laser Doppler flowmetry method. The μHematron device is dedicated to the non-invasive measurement of effective thermal conductivity of living tissues, based on the thermal clearance method. Its major advantage is its ambulatory functionality, as available methods for evaluation of microcirculatory activity are non-ambulatory methods. An experiment was conducted on ten healthy women exposed for one hour in three different thermal environments (22 °C, 25 °C and 30 °C). Skin microcirculatory activity was analyzed after an acclimatization period of 30 minutes. The time between each exposure was at least one hour. Performances of the μHematron device were assessed and a comparative study with a laser Doppler perfusion monitor (LDPM) was performed. Good correlation coefficients between the two devices (r = 0.71 at T1 = 22 °C, r = 0.77 at T2 = 25 °C and r = 0.83 at T3 = 30 °C) were obtained while the LDPM signal was filtered by a low pass filter (0.1 Hz). These results showed that continuous monitoring of effective thermal conductivity was possible in neutral and warm ambiences. Then, the μHematron device could be considered as a complementary tool to Doppler techniques for the investigation of skin blood flow, when ambulatory conditions are required.     

AhpC (alkyl hydroperoxide reductase) from Anabaena sp. PCC 7120 protects Escherichia coli from multiple abiotic stresses

Alkyl hydroperoxide reductase (AhpC) is known to detoxify peroxides and reactive sulfur species (RSS). However, the relationship between its expression and combating of abiotic stresses is still not clear. To investigate this relationship, the genes encoding the alkyl hydroperoxide reductase (ahpC) from Anabaena sp. PCC 7120 were introduced into E. coli using pGEX-5X-2 vector and their possible functions against heat, salt, carbofuron, cadmium, copper and UV-B were analyzed. The transformed E. coli cells registered significantly increase in growth than the control cells under temperature (47 °C), NaCl (6% w/v), carbofuron (0.025 mg ml^?1), CdCl₂ (4 mM), CuCl₂ (1 mM), and UV-B (10 min) exposure. Enhanced expression of ahpC gene as measured by semi-quantitative RT-PCR under aforementioned stresses at different time points demonstrated its role in offering tolerance against multiple abiotic stresses.     

Rapid in vitro micropropagation of Agapanthus praecox

In vitro micropropagation and acclimatization for the ornamental Agapanthus praecox, are reported. The influence of different growth regulators on shoot multiplication from shoot-tip explants of A. praecox was investigated. Prolific shoot multiplication (47.3 ± 1.96 shoots per explant) was achieved on Murashige and Skoog (MS) medium supplemented with 22.2 μM benzyladenine (BA), 2.9 μM indole-3-acetic acid (IAA), and 4.5 μM thidiazuron (TDZ). Shoots were rooted on half-strength MS basal medium supplemented with 5.7 μM IAA and 2.5 μM 2-isopentenyladenine (2iP) with 11.3 ± 0.78 roots per shoot. The in vitro-raised plants were established successfully in a 1:1 (v/v) vermiculite:sand mixture when maintained in a greenhouse with 100% survival. The elongated shoots (more than 5 cm in length) were treated for rooting and acclimatization in a moistened (5.7 μM IAA and 2.5 μM 2iP) vermiculite:sand (1:1 v/v) mixture, first in the misthouse and then in the greenhouse. Rooting and acclimatization was achieved simultaneously (100%) in the misthouse which was followed by greenhouse cultivation. This system can be used for rapid mass clonal propagation of A. praecox, for conservation strategies, commercial production, gene transformation studies and to produce phytomedicines.     

Culture filtrate of root endophytic fungus Piriformospora indica promotes the growth and lignan production of Linum album hairy root cultures

The effect of addition of autoclaved and filter-sterilized culture filtrate of Piriformospora indica (a root endophytic fungus) to the growing Linum album hairy root cultures on growth and lignan production was investigated. The addition resulted in a significant enhancement in lignan production and growth. The podophyllotoxin and 6-methoxypodophyllotoxin (the lignans) concentrations were maximally improved by 3.8 times (233.8 mg/L) and 4.4 times (131.9 mg/L) in comparison to control cultures, respectively, upon addition of 3.0% (v/v) filter-sterilized culture filtrate of P. indica to the hairy root cultures of L. album for exposure time of 48 h. This increase in the lignan content also coincided with the increase in phenylalanine ammonia lyase activity, which was 3.1-fold (371.4 μkat/kg protein) higher compared to control cultures under the same conditions. The maximal increase in hairy root biomass was, however, obtained under different conditions; it was enhanced by 1.4 times (21.8 g/L) in comparison to control cultures, when 2% (v/v) filter-sterilized culture filtrate was in contact with L. album cultures for 96 h.     

Cryopreservation of kangaroo spermatozoa using alternative approaches that reduce cytotoxic exposure to glycerol

Alternative techniques for the cryopreservation of kangaroo spermatozoa that reduced or eliminated the need for glycerol were investigated including; (1) freezing spermatozoa with 20% glycerol in pre-packaged 0.25 mL Cassou straws to enable rapid dilution of the glycerol post-thaw, (2) investigating the efficacy of 20% (v/v) dimethyl sulphoxide (DMSO) and dimethylacetamide (DMA—10%, 15% and 20% v/v) as cryoprotectants and (3) vitrification of spermatozoa with or without cryoprotectant (20% v/v glycerol, 20% v/v DMSO and 20% v/v DMA). Immediate in-straw post-thaw dilution of 20% glycerol and cryopreservation of spermatozoa in 20% DMSO produced no significant improvement in post-thaw viability of kangaroo spermatozoa. Spermatozoa frozen in 20% DMA showed post-thaw motility and plasma membrane integrity of 12.7 ± 1.9% and 22.7 ± 5.4%, respectively, while kangaroo spermatozoa frozen by ultra-rapid freezing techniques showed no evidence of post-thaw viability. The use of 10–20% DMA represents a modest but significant improvement in the development of a sperm cryopreservation procedure for kangaroos.