Extraction of chlorophyll a from freshwater phytoplankton for spectrophotometric analysis

The efficiency of pigment extraction forms the crux of the spectrophotometric analysis of chlorophyll a. The alcoholic solvents, methanol and ethanol, proved to be superior to acetone and acetone with DMSO. Homogenisation and sonication did not improve the extraction in the alcoholic solvents. Boiling at 100°C had an adverse effect whereas complete extraction of the pigments was obtained at the solvents boiling point and allowing the samples to stand for 24 h in the dark.     

Cryopreservation of the Mediterranean mussel (Mytilus galloprovincialis) spermatozoa

The effects of cryoprotectants, cooling rate and freezing on the mussel Mytilus galloprovincialis sperm were evaluated. At the end of each step of the experimental protocol, motility and fertilization ability of sperm were analyzed, compared to fresh semen. Five cryoprotectants were tested in their toxicity level: dimethylsulfoxide, ethylene glycol, 1-2 propylene glycol at 5%, 7%, 10%, 15% and 20% concentration; glycerol and methanol at concentration of 5%, 7% and 10%. The incubation times were 10, 20 and 30 min at 20 ± 1 °C. Only dimethylsulfoxide, ethylene glycol and 1-2 propylene glycol at 5%, 7% and 10% were chosen for the following pre-freezing step. Five adaptation/chilling rates were analyzed: 10 min at 20 ± 1, −2, −1, −0.5 and −0.25 °C/min and the last one was used for testing the best freezing procedure among seven gradients. Particularly, two rapid rates, three slow rates and two double step rates were conducted.Thawing results showed that M. galloprovincialis sperm are very sensitive to rapid pre-freezing and freezing protocols and only a slow procedure assured good motility and fertilization percentages.     

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.     

Membrane hydration correlates to cellular biophysics during freezing in mammalian cells

Cell survival during freezing applications in biomedicine is highly correlated to the temperature history and its dependent cellular biophysical events of dehydration and intracellular ice formation (IIF). Although cell membranes are known to play a significant role in cell injury, a clear correlation between the membrane state and the surrounding intracellular and extracellular water is still lacking. We previously showed that lipid hydration in LNCaP tumor cells is related to cellular dehydration. The goal of this study is to build upon this work by correlating both the phase state of the membrane and the surrounding water to cellular biophysical events in three different mammalian cell types: human prostate tumor cells (LNCaP), human dermal fibroblasts (HDF), and porcine smooth muscle cells (SMC) using Fourier Transform Infrared spectroscopy (FTIR). Variable cooling rates were achieved by controlling the degree of supercooling prior to ice nucleation (− 3 °C and − 10 °C) while the sample was cooled at a set rate of 2 °C/min. Membranes displayed a highly cooperative phase transition under dehydrating conditions (i.e. NT = − 3 °C), which was not observed under IIF conditions (NT = − 10 °C). Spectral analysis showed a consistently greater amount of ice formation during dehydrating vs. IIF conditions in all cell types. This is hypothesized to be due to the extreme loss of membrane hydration in dehydrating cells that is manifested as excess water available for phase change. Interestingly, changes in residual membrane conformational disorder correlate strongly with cellular volumetric decreases as assessed by cryomicroscopy. A strong correlation was also found between the activation energies for freezing induced lyotropic membrane phase change determined using FTIR and the water transport measured by cryomicroscopy. Reduced lipid hydration under dehydration freezing conditions is suggested as one of the likely causes of what has been termed as “solution effects” injury in cryobiology.     

A method for enzyme quenching in microbial metabolome analysis successfully applied to gram-positive and gram-negative bacteria and yeast

Although microbial metabolome analysis has now become a widely used method, no generally applicable quenching method has been published so far. Either the methods were established for only one defined organism or the metabolite coverage was quite low. In the current work, a novel, reliable, and robust quenching method for different types of organisms is described. Compared with the commonly used quenching procedure with 60% methanol (−50 °C), we obtained improved results for three examined organisms with different cell wall and membrane structures using a 40% ethanol/0.8% sodium chloride solution (−20 °C). Increased metabolite levels were achieved for 60-80% of all identified compounds. Moreover, the estimated standard error of the relative concentrations of 120-160 different substances was only 14 ± 4% compared with 17 ± 3% in unquenched samples and 24 ± 7% in samples quenched with methanol for the different tested organisms.     

Cold tolerance and freeze-induced glucose accumulation in three terrestrial slugs

Cold tolerance and metabolic responses to freezing of three slug species common in Scandinavia (Arion ater, Arion rufus and Arion lusitanicus) are reported. Autumn collected slugs were cold acclimated in the laboratory and subjected to freezing conditions simulating likely winter temperatures in their habitat. Slugs spontaneously froze at about − 4 °C when cooled under dry conditions, but freezing of body fluids was readily induced at − 1 °C when in contact with external ice crystals. All three species survived freezing for 2 days at − 1 °C, and some A. rufus and A. lusitanicus also survived freezing at − 2 °C. ¹H NMR spectroscopy revealed that freezing of body fluids resulted in accumulation of lactate, succinate and glucose. Accumulation of lactate and succinate indicates that ATP production occurred via fermentative pathways, which is likely a result of oxygen depletion in frozen tissues. Glucose increased from about 6 to 22 μg/mg dry tissue upon freezing in A. rufus, but less so in A. ater and A. lusitanicus. Glucose may thus act as a cryoprotectant in these slugs, although the concentrations are not as high as reported for other freeze tolerant invertebrates.     

Investigating sperm cryopreservation in a model tunicate, Ciona intestinalis sp. A

In cryopreservation procedures, the capacity to protect the cells from freezing and thawing processes is sensitive to the choice of the cryoprotective agent (CPA) and to its optimal concentration. The advancement of research on Tunicate model species has raised interest in liquid nitrogen cryopreservation for the storage and distribution of genetic resources. Ciona intestinalis (Linnè, 1767) consists of a complex of cryptic taxa that are central to several areas of investigation, from comparative genomics to invasive biology. Here we investigated how five CPAs, three chilling rates and two freezing rates influence semen cryopreservation in C. intestinalis sp. A. By using larval morphology and motility as endpoints, we estimated that long term semen storage requires 10% dimethyl sulfoxide as a protective agent, −1 °C/min chilling rate (18 °C to 5 °C) and −13 °C/min freezing rate (5 °C to −80 °C), followed by immersion in liquid nitrogen.     

Survival of Pacific oyster, Crassostrea gigas, oocytes in relation to intracellular ice formation

The effect of IIF in Pacific oyster oocytes was studied using cryo and transmission electron microscopy (TEM). The viability of oocytes at each step of a published cryopreservation protocol was assessed in an initial experiment. Two major viability losses were identified; one when oocytes were cooled to −35 °C and the other when oocytes were plunged in liquid nitrogen. Although the cryomicroscope showed no evidence of IIF in oocytes cooled with this protocol, TEM revealed that these oocytes contained ice crystals and were at two developmental stages when frozen, prophase and metaphase I. To reduce IIF, the effect of seven cooling programmes involving cooling to −35 or −60 °C at 0.1 or 0.3 °C min⁻¹ and holding for 0 or 30 min at −35 or −60 °C was evaluated on post-thaw fertilization rate of oocytes. Regardless of the cooling rate or holding time, the fertilization rate of oocytes cooled to −60 °C was significantly lower than that of oocytes cooled to −35 °C. The overall results indicated that observations of IIF obtained from cryomicroscopy are limited to detection of larger amounts of ice within the cells. Although the amount of cellular ice may have been reduced by one of the programmes, fertilization was reduced significantly; suggesting that there is no correlation between the presence of intracellular ice and post-thaw fertilization rate. Therefore, oyster oocytes may be more susceptible to the effect of high solute concentrations and cell shrinkage than intracellular ice under the studied conditions.     

Cryopreservation of collared peccary (Pecari tajacu) semen using different freezing curves,straw sizes,and thawing rates

The objective of this study was to verify the effect of different freezing curves, straw sizes, and thawing rates on the cryopreservation of collared peccary semen. Twelve ejaculates were obtained from captive adult males by electroejaculation, and evaluated for sperm motility, kinetic rating, viability, morphology, and functional membrane integrity. The ejaculates were diluted in a coconut water extender (ACP-116c) with egg yolk and glycerol, packaged into 0.25 mL or 0.50 mL plastic straws and cryopreserved in liquid nitrogen following a slow (−10 °C/min) or a fast (−40 °C/min) freezing curve. After one week, samples were thawed at 37 °C/1 min or 70 °C/8 s and evaluated as reported for fresh semen, and also for kinematic parameters (computerized analysis). A significant decrease in sperm motility and kinetic rating was observed after glycerol addition at 5 °C and also after thawing for all the treatments (P < 0.05). Regarding post-thaw semen variables, no differences were verified between freezing curves when the same straw size and thawing rate were taken as reference (P > 0.05). In general, values for sperm characteristics found after thawing at 37 °C were better preserved than at 70 °C (P < 0.05), both in the use of 0.25 mL or 0.50 mL straws, which were similar for semen packaging (P > 0.05). The evaluation of the kinematic parameters of sperm motility confirmed these results at values varying from 20% to 30% motile sperm for the samples thawed at 37 °C, and values fewer than 12% motile sperm for samples thawed at 70 °C (P < 0.05). In conclusion, we recommend the use of a fast freezing curve that reduces the time spent on the cryopreservation of collared peccary semen, which could be packaged both in 0.25 mL or 0.50 mL straws, but the thawing should be conducted at 37 °C/1 min.     

Cooling rate optimization for zebrafish sperm cryopreservation using a cryomicroscope coupled with SYBR14/PI dual staining

The Zebrafish has gained increased popularity as an aquatic model species in various research fields, and its widespread use has led to numerous mutant strains and transgenic lines. This creates the need to store these important genetic materials as frozen gametes. Sperm cryopreservation in zebrafish has been shown to yield very low post-thaw survival and many protocols suffer from great variability and poor reproducibility. The present study was intended to develop a freezing protocol that can be reliably used to cryopreserve zebrafish sperm with high post-thaw survival. In particular, our study focused on cooling protocol optimization with the aid of cryomicroscopy. Specifically, sperm suspended in 8% DMSO or 4% MeOH were first incubated with live/dead fluorescent dyes (SYBR14/PI) and then cooled at various rates from 4 °C to different intermediate stopping temperatures such as −10, −20, −30 and −80 °C before rewarming to 35 °C at the rate of 100 °C/min. %PI-positive (dead) cells were monitored throughout the cooling process and this screening yielded an optimal rate of 25 °C/min for this initial phase of freezing. We then tested the optimal cooling rate for the second phase of freezing from various intermediate stopping temperatures to −80 °C before plunging into liquid nitrogen. Our finding yielded an optimal intermediate stopping temperature of −30 °C and an optimal rate of 5 °C/min for this second phase of freezing. When we further applied this two-step cooling protocol to the conventional controlled-rate freezer, the average post-thaw motility measured by CASA was 46.8 ± 6.40% across 11 males, indicating high post-thaw survival and consistent results among different individuals. Our study indicates that cryomiscroscopy is a powerful tool to devise the optimal cooling conditions for species with sperm that are very sensitive to cryodamage.     

Cell disruption using a different methodology for proteomics analysis of Trypanosoma cruzi strains

We have developed a cell disruption method to produce a protein extract using Trypanosoma cruzi cells based on a straightforward hypoosmotic lysis protocol. The procedure consists of three steps: incubation of the cells in a hypoosmotic lysis buffer, sonication in a water bath, and centrifugation. The final protein extract was designated TcS12. The stages of cell disruption at different incubation times were monitored by differential interference contrast microscopy. After 30 min of incubation in lysis buffer at 4 °C, the T. cruzi epimastigote forms changed from slender to round-shaped parasites. Nevertheless, cell disruption took place following sonication of the sample for 30 min. The efficiency of the methodology was also validated by flow cytometry, which resulted in 72% of propidium iodide (PI)-labeled cells. To estimate the protein extraction yield and the differential protein expression, the proteomics profile of four T. cruzi strains (CL-Brener, Dm28c, Y, and 4167) were analyzed by liquid chromatography tandem mass spectrometry (LCMS/MS) on a SYNAPT HDMS system using the label-free MS^E approach. ProteinLynx Global Server (version 2.5) with Expression^E analysis identified a total of 1153 proteins and revealed 428 differentially expressed proteins among the strains. Gene ontology analysis showed that not only cytosolic proteins but also nuclear and organellar ones were present in the extract.     

Comparative cryopreservation study of trochophore larvae from two species of bivalves: Pacific oyster (Crassostrea gigas) and Blue mussel (Mytilus galloprovincialis)

Oysters and mussels are among the most farmed species in aquaculture industry around the world. The aim of this study was to test the toxicity of cryoprotective agents to trochophore larvae from two different species of bivalves and develop an improved cryopreservation protocol to ensure greater efficiency in the development of cryopreserved trochophores (14 h old oyster larvae and 20 h old mussel larvae) to normal D-larvae for future developments of hatchery spat production. The cryopreservation protocol producing the best results for oyster trochophores (60.0 ± 6.7% normal D-larvae) was obtained by holding at 0 °C for 5 min then cooling at 1 °C min⁻¹ to −10 °C and holding for 5 min before cooling at 0.5 °C to −35 °C, holding 5 min and then plunging into liquid nitrogen (LN), using 10% ethylene glycol. For mussel experiments, no significant differences were found when cooling at 0.5 °C min⁻¹ or at 1 °C min⁻¹ for CPA combinations with 10% ethylene glycol and at 0.5 °C min⁻¹. Using these combinations, around half of trochophores were able to develop to normal D-larvae post-thawing (48.9 ± 7.6% normal D-larvae).     

Plant protein isolation and stabilization for enhanced resolution of two-dimensional polyacrylamide gel electrophoresis

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the common method of choice for proteomic analysis. By introducing several small changes, a method was developed that not only improved the resolution and reproducibility of 2D-PAGE but also shortened the time of analysis. Precipitation by alkaline phenol and methanol/ammonium acetate was the choice for protein extraction. However, instead of precipitating the proteins overnight at -20 °C, it was carried out for 2 to 3 h at -80 °C. Ethanol was used for the final wash of the protein precipitate instead of routinely used acetone. Dithiothreitol (DTT) was used in all solutions from the beginning, considerably improving the solubilization of precipitated proteins. Solubilization was further improved by using a mixture of detergents and denaturants at high concentrations along with large amounts of DTT. Both in-gel rehydration and cup-loading methods were used for isoelectric focusing (IEF). For in-gel rehydration, samples reduced with DTT were diluted with sample buffer containing 2-hydroxyethyl disulfide (2-HED) (1:3) or were cup-loaded on a strip rehydrated with sample buffer containing 2-HED. Glycerol (5%) was used in the sample buffer, and the focusing was performed at 15 °C. The applicability of the method was demonstrated using several soybean tissues.     

Effects of acclimation and diapause on the thermal tolerance of the two-spotted spider mite Tetranychus urticae

The two-spotted spider mite, Tetranychus urticae, is a worldwide pest species that overwinters as diapausing females. Cold hardening is presumed to start during diapause development to ensure the successful overwintering of this species. To address this hypothesis, we compared cold tolerance between non-diapausing and diapausing females. We measured supercooling point (SCP) and survival to acute cold stress by exposing the mites at a range of sub-zero temperatures (from −4 to −28 °C for 2 h). The mean SCPs of non-diapausing and diapausing females were −19.6±0.5 and −24.7±0.3 °C respectively, and freezing killed the mites. Diapausing females were significantly more cold tolerant than non-diapausing ones, with LT₅₀ of −19.7 and −13.3 °C, respectively. Further, we also examined the effects of cold acclimation (10 d at 0 or 5 °C) in non-diapausing and diapausing females. Our findings indicated that diapause decreased SCP significantly, while cold acclimation had no effect on the SCP except for non-diapausing females that were acclimated at 5 °C. Acclimation at 5 °C enhanced survival to acute cold stress in diapausing and non-diapausing females, with LT₅₀ of −22.0 and −17.1 °C, respectively. Altogether, our results indicate that T. urticae is a chill tolerant species, and that diapause and cold acclimation elevate cold hardiness in this species.     

Mycosporine-like amino acids in azooxanthellate and zooxanthellate early developmental stages of the soft coral Heteroxenia fuscescens

The ontogenetic changes of MAAs in the soft coral Heteroxenia fuscescens was studied in relation to their symbiotic state (azooxanthellate vs. zooxanthellate) under different temperature conditions in the Gulf of Eilat, northern Red Sea. The HPLC chromatograms for extracts of the planulae, azoo- and zooxanthellate primary polyps of H. fuscescens from all dates of collection yielded a single peak at 320 nm that has been identified as the compound palythine. Concentration of palythine in planulae at 23 °C was 7.57 ± 1 nmol mg^− 1 protein and at 28 °C reached 17.29 ± 1 nmol × mg^− 1 protein. Concentration of palythine in azooxanthellate primary polyps was 16.4 ± 3 nmol × mg^− 1 protein and 28.37 ± 2.8 nmol × mg^− 1 protein at 23 °C and 28 °C respectively. The palythine concentration for zooxanthellate primary polyps at 23 °C was 13 ± 3 nmol × mg^− 1 protein and at 28 °C 32.7 ± 2 nmol mg^− 1 protein. Palythine concentrations were significantly higher at 28 °C in the different animal groups and correlated linearly with the ambient collection temperature. This study shows for the first time that UVR and temperature act synergistically and affect the MAA levels of early life-history stages of soft corals.     

Analysis of skeletal muscle metabolome: Evaluation of extraction methods for targeted metabolite quantification using liquid chromatography tandem mass spectrometry

Functional metabolomics of skeletal muscle involves the simultaneous identification and quantification of a large number of metabolites. For this purpose, the extraction of metabolites from animal tissues is a crucial technical step that needs to be optimized. In this work, five extraction methods for skeletal muscle metabolome analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS) were tested. Bird skeletal muscles sampled postmortem and quenched in liquid nitrogen were used. Three replicates of the same sample were extracted using the following solvent systems of varying polarity: boiling water (BW, +100 °C), cold pure methanol (CPM, −80 °C), methanol/chloroform/water (MCW, −20 °C), boiling ethanol (BE, +80 °C), and perchloric acid (PCA, −20 °C). Three injections by extraction were performed. The BW extraction showed the highest recovery of metabolites with the lowest variability (<10%) except for creatine-phosphate (creatine-P). Considering yield (area of the peaks), reproducibility, and ease, the current experiment drew a scale for the muscle metabolome extraction starting from the best to the least convenient: BW > MCW > CPM > PCA ? BE. In addition, the semiquantification of metabolites in two muscles showing different metabolic and contractile properties was carried out after BW extraction and showed expected differences in metabolite contents, thereby validating the technique for biological investigations. In conclusion, the BW extraction is recommended for analysis of skeletal muscle metabolome except for creatine-P, which was poorly recovered with this technique.     

Cryopreservation of Greenshell™ mussel (Perna canaliculus) trochophore larvae

The Greenshell™ mussel (Perna canaliculus) is the main shellfish species farmed in New Zealand. The aim of this study was to evaluate the effects of cryoprotectant concentration, loading and unloading strategy as well as freezing and thawing method in order to develop a protocol for cryopreservation of trochophore larvae (16–20 h old). Toxicity tests showed that levels of 10–15% ethylene glycol (EG) were not toxic to larvae and could be loaded and unloaded in a single step. Through cryopreservation experiments, we designed a cryopreservation protocol that enabled 40–60% of trochophores to develop to D-larvae when normalized to controls. The protocol involved: holding at 0 °C for 5 min, then cooling at 1 °C min⁻¹ to −10 °C, holding for a further 5 min, then cooling at 0.5 °C min⁻¹ to −35 °C followed by a 5 min hold and then plunging into liquid nitrogen. A final larval rearing experiment of 18 days was conducted to assess the ability of these frozen larvae to develop further. Results showed that only 2.8% of the frozen trochophores were able to develop to competent pediveligers.     

Viability of fastidious Phytophthora following different cryopreservation treatments

Living stock cultures with constant phenotypes and genotypes are required for a wide range of research and industrial applications; however, long-term, stable preservation of fastidious Phytophthora strains has been challenging. In this study, we systematically evaluated different cryopreservation treatments to identify and clarify freezing, thawing, and other conditions appropriate for long-term maintenance. Optimal preservation conditions were largely strain-specific, with robust strains remaining fully viable and the fastidious yielding lower recovery under all test conditions. Nevertheless, several procedures were shown to be generally applicable for effective cryopreservation of most Phytophthora organisms. Fastidious strains retained higher viability following the −1 °C min⁻¹ freezing protocol (Mr Frosty's) than either of two widely used programmed freezing procedures. Revival was higher when frozen mycelium plugs were thawed at 37 °C for 2 min or 25 °C for 5 min, while lower viability was apparent for fastidious strains thawed at 55 °C for 1.5 min. Among 15 cryoprotective solutions assessed, 5 % dimethyl sulfoxide produced the highest viability for all fastidious strains. The effect of prefreeze and postfreeze treatments on revival was mild, if any, and strain-dependent. This study has generated reliable, practical, long-term preservation solutions applicable to a majority of Phytophthora species. It also has revealed a need for in-depth physiological and morphological investigations to further enhance the preservation methods for fastidious strains.     

Development of cryopreservation protocols for early stage zebrafish (Danio rerio) ovarian follicles using controlled slow cooling

Cryopreservation of germplasm of aquatic species offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful fish sperm cryopreservation has been achieved with many species, there has been no report of successful cryopreservation of fish embryos and late stage oocytes which are large, chilling sensitive and have low membrane permeability. In the present study, cryopreservation of early stage zebrafish ovarian follicles was studied for the first time using controlled slow freezing. The effect of cryoprotectant, freezing medium, cooling rate, method for cryoprotectant removal, post-thaw incubation time and ovarian follicle developmental stage were investigated. Stages I and II ovarian follicles were frozen in 4 M methanol and 3 M DMSO in either L-15 medium or KCl buffer. Ovarian follicle viability was assessed using trypan blue, FDA + PI staining and ADP/ATP assay. The results showed that KCl buffer was more beneficial than L-15 medium, methanol was more effective than DMSO, optimum cooling rates were 2-4 °C/min, stepwise removal of cryoprotectant improved ovarian follicle viability significantly and stage I ovarian follicles were more sensitive to freezing. The results also showed that FDA + PI staining and ADP/ATP assay were more sensitive than TB staining. The highest follicle viabilities after post-thaw incubation for 2 h obtained with FDA + PI staining were 50.7 ± 4.0% although ADP/ATP ratios of the cryopreserved follicles were significantly increased indicating increased cell damage. Studies are currently being carried out on in vitro maturation of these cryopreserved ovarian follicles.     

Adverse effect of cake collapse on the functional integrity of freeze-dried bull spermatozoa

Under optimal freeze-drying conditions, solutions exhibit a cake-like porous structure. However, if the solution temperature is higher than the glass transition temperature of the maximally freeze-concentrated phase (T_g′) during drying phase, the glassy matrix undergoes viscous flow, resulting in cake collapse. The purpose of the present study was to investigate the effect of cake collapse on the integrity of freeze-dried bull spermatozoa. In a preliminary experiment, factors affecting the T_g′ of conventional EGTA buffer (consisting of Tris–HCl, EGTA and NaCl) were investigated in order to establish the main experimental protocol because EGTA buffer T_g′ was too low (−45.0 °C) to suppress collapse. Modification of the EGTA buffer composition by complete removal of NaCl and addition of trehalose (mEGTA buffer) resulted in an increase of T_g′ up to −27.7 °C. In the main experiment, blastocyst yields after ooplasmic injection of freeze-dried sperm preserved in collapsed cakes (drying temperature: 0 or −15 °C) were significantly lower than those of sperm preserved in non-collapsed cake (drying temperature: −30 °C). In conclusion, freeze-dried cake collapse may be undesirable for maintaining sperm functions to support embryonic development, and can be inhibited by controlling both T_g′ of freeze-drying buffer and temperature during the drying phase.