Short-term storage of ova of common carp and tench in extenders

In a study of the effect of short-term storage on the hatching rate of common carp Cyprinus carpio and tench Tinca tinca ova in vitro in various extenders at 21° C under aerobic conditions, the best extender for 30 min storage for common carp appeared to be Dettlaff 1. This gave the same hatching rate as controls without extender (55% v. 56%). For 60 min storage of ova, the best extenders were Dettlaff 2 (24% hatching rate) and Dettlaff 3 (30%), but hatching was significantly lower than in the control (58%). In carp ovarian artificial fluid (CAF) extender, the hatching rate of common carp ova was also high after 10 min, but decreased to 12% after 30 min. In tench, the hatching rate of ova increased after 10 min storage in Dettlaff 5 extender (44%) compared to the control (41%) without extender. However, it was significantly lower after storage in Dettlaff 1, 2, 3, 4, 5 and CAF extenders for 20, 30 and 60 min, compared to controls. Malformations (10–50%) were observed in the tench second control groups without extender after 10, 20 and 30 min storage of ova.     

Plant-microbe competition for soil amino acids in the alpine tundra: effects of freeze-thaw and dry-rewet events

Amino acids have been shown to be a potentially significant N source for the alpine sedge, Kobresia myosuroides. We hypothesised that freeze-thaw and dry-rewet events allow this plant species increased access to amino acids by disrupting microbial cells, which decreases the size of competing microbial populations, but increases soil amino acid concentrations. To test this hypothesis, we characterized freeze-thaw and dry-rewet events in the field and simulated them in laboratory experiments on plant-soil microcosms. In one experiment, ¹⁵N,¹³C-[2]-glycine was added to microcosms that had previously been subjected to a freeze-thaw or dry-rewet event, and isotopic concentrations in the plant and microbial fractions were compared to non-stressed controls. Microbial biomass and uptake of the labeled glycine were unaffected by the freezing and drying treatments, but microbial uptake of ¹⁵N was lower in the two warmer treatments (dry-rewet and summer control) then in the two colder treatments (freeze-thaw and fall control). Plant uptake of glycine-¹⁵N was decreased by climatic disturbance, and uptake in plants that had been frozen appeared to be dependent on the severity of the freeze. The fact that intact glycine was absorbed by the plants was confirmed by near equal enrichment of plant tissues in ¹³C and ¹⁵N. Plants under optimal conditions recovered 3.5% of the added ¹⁵N and microbes recovered 5.0%. The majority of the ¹³C and ¹⁵N label remained in a non-extractable fraction in the bulk soil. To better understand the isolated influences of environmental perturbations on soil amino acid pools and population sizes of amino-acid utilizing microbes, separate experiments were performed in which soils, alone, were subjected to drying and rewetting or freezing and thawing. Potential respiration of glycine and glutamate (substrate-induced respiration; SIR) by the soil microbial communities was unaffected by a single freeze-thaw event. Glycine SIR was decreased slightly (∼10%) by the most extreme drying treatment, but glutamate SIR was not significantly affected. Freezing lowered the concentration of water-extractable amino acids while drying increased their concentration. We interpret the surprising former result as either a decrease in proteolytic activity in frozen soils relative to amino acid uptake, or a stimulation in microbial uptake by physical nutrient release from the soil. We conclude that climatic disturbance does not provide opportunities for increased amino acid uptake by K. myosuroides, but that this plant competes well for amino acid N under non-stressed conditions, especially when soils are warm. We also note that this alpine tundra microbial community's high resistance to freeze-thaw and dry-rewet events is novel and contrasts with studies in other ecosystems. Received: 24 February 1997 / Accepted: 28 August 1997     

Cryopreservation produces limited long-term effects on the nematode Caenorhabditis elegans

Cryopreservation, the freezing and later warming of biological samples with minimal loss of viability, is important in many scientific disciplines. For some applications, particularly those where there is limited available material, it is critical to ensure the maximal survival rates of cryopreserved materials. Most of the challenges encountered with such techniques take place after the warming process where cryodamage affects cell viability and future development. Here we have used the nematode Caenorhabditis elegans to investigate the effects of cryodamage caused by slow-freezing. We find that freezing results in the death of some worms, with an approximately 40% reduction in the number of worms that develop in the frozen populations, but that the effects on worms that survive are limited. For example, there are no differences in the lifetime fecundity or in lifespan between frozen and control worms, although early fecundity and body size was reduced in frozen worms. Similarly, analyses of body wall muscle structure and of pharyngeal function indicates that muscle development and function are not significantly affected by freezing. We do however determine that freezing increases the rates of matricidal hatching, where progeny hatch within the mother. Overall, these results indicate that, for worms that survive, cryopreservation produces limited long-term effects, but do indicate that some phenotypes could be used in further analyses of the cellular damage induced by cryopreservation.     

Ice-active proteins and cryoprotectants from the New Zealand alpine cockroach, Celatoblatta quinquemaculata

Celatoblatta quinquemaculata is moderately freezing tolerant. We have investigated low and high molecular weight compounds that may be associated with its survival. Glycerol and trehalose were identified as potential cryoprotectants, with trehalose at the higher concentration. Trehalose was at its highest concentration in late autumn, during the periods sampled. Water contents declined with time and were significantly lower in late autumn than in late summer. No thermal hysteresis activity was detected in haemolymph or in extracts of the head, muscles and the fat body. Extracts of the Malpighian tubules showed an hexagonal crystal growth form, as did those of the gut tissue and gut contents. The gut tissue had high levels of thermal hysteresis (∼2 °C) and the gut contents somewhat lower levels (∼0.6 °C). Recrystallization inhibition activity mirrored that of thermal hysteresis, with activity absent in the haemolymph or fat body cells but present in the gut tissues and contents. Activity was reduced by heating and was associated with a molecule >14 kDa in size. These findings suggest an antifreeze protein is involved. In fed animals, ice nucleation is likely to start in the gut. Gut cells have a much greater resistance to freezing than do fat body or Malpighian tubule cells. The antifreeze protein may enable this tissue to survive freezing stress by inhibiting recrystallization.     

Preservation of bovine preantral follicle viability and ultra-structure after cooling and freezing of ovarian tissue

Bovine preantral follicles within ovarian fragments were exposed and cryopreserved in absence or presence of 1.5 M glycerol (GLY), ethylene glycol (EG), propanediol (PROH) or dimethyl sulfoxide (DMSO), undergoing a previous cooling at 20 °C for 1 h (protocol 1) or at 4 °C for 24 h (protocol 2) in 0.9% saline solution. At the end of each treatment, preantral follicles were classified as non-viable/viable when they were stained/not stained with trypan blue, respectively. To confirm viability staining, ultra-structure of the follicles was evaluated by transmission electronic microscopy (TEM). Data were compared by Chi-square test (P < 0.05). The storage of the ovaries at 20 °C for 1 h (78%) and 4 °C for 24 h (80%) did not reduce significantly the percentage of viable preantral follicles when compared to the control (75%). Similar results were obtained when ovarian fragments, respectively, for protocols 1 and 2, were exposed to MEM (78 and 77%), 1.5 M EG (78 and 71%), as well as frozen in 1.5 M EG (74 and 77%). Percentages of viable follicles in control were similar to those observed after exposure (75%) and freezing (76%) in presence of 1.5 M DMSO only when protocol 1 was used. The increase of the concentration from 1.5 to 3.0 M, for all cryoprotectants, reduced significantly the percentage of viable preantral follicles after freezing. Ultra-structural analysis has confirmed trypan blue results, showing that not only basement membrane, but also organelles, were intact in viable preantral follicles. In conclusion, ovarian tissue cooling at 4 °C for 24 h before cryopreservation (protocol 2) does not affect the viability of bovine preantral follicles when 1.5 M EG is present in the cryopreservation medium.     

Quality of frozen-thawed semen in brown bear is not affected by timing of glycerol addition

We have tested several freezing protocols for brown bear semen, modifying the time when glycerol was added (before and after cooling to 5 °C). No differences were found among protocols, indicating a good tolerance of brown bear semen to glycerol. This finding indicates that freezing protocols for brown bear semen could be modified to fit practical solutions which would facilitate preparation of the seminal samples in the field with the addition of glycerol at ambient temperature.     

Stainless steel tube-based cell cryopreservation containers

This study focused on increasing the freezing rate in cell vitrification cryopreservation by using a cryopreservation container possessing rigid mechanical properties and high heat-transfer efficiency. Applying a fast freezing rate in vitrification cryopreservation causes a rapid temperature change in the cryopreservation container and has a substantial impact on mechanical properties; therefore, a highly rigid cryopreservation container that possesses a fast freezing rate must be developed. To produce a highly rigid cryopreservation container possessing superior heat transfer efficiency, this study applies an electrochemical machining (ECM) method to an ANSI 316L stainless steel tube to treat the surface material by polishing and roughening, thereby increasing the freezing rate and reducing the probability of ice crystal formation. The results indicated that the ECM method provided high-quality surface treatment of the stainless steel tube. This method can reduce internal surface roughness in the stainless steel tube, thereby reducing the probability of ice crystal formation, and increase external surface roughness, consequently raising convection heat-transfer efficiency. In addition, by thinning the stainless steel tube, this method reduces heat capacity and thermal resistance, thereby increasing the freezing rate. The freezing rate (3399 ± 197 °C/min) of a stainless steel tube after interior and exterior polishing and exterior etching by applying ECM compared with the freezing rate (1818 ± 54 °C/min) of an original stainless steel tube was increased by 87%, which also exceeds the freezing rate (2015 ± 49 °C/min) of an original quartz tube that has a 20% lower heat capacity. However, the results indicated that increasing heat-transferring surface areas and reducing heat capacities cannot effectively increase the freezing rate of a stainless steel tube if only one method is applied; instead, both techniques must be implemented concurrently to improve the freezing rate.     

Optimizing conditions for treating goat semen with cholesterol-loaded cyclodextrins prior to freezing to improve cryosurvival

The fertility of goat sperm is highly variable and new methods for improving sperm cryosurvival are needed. Cholesterol plays important roles in membrane fluidity, cold shock sensitivity and cryodamage, and treating sperm from cold-shock sensitive species with cholesterol-loaded cyclodextrins (CLC) prior to cryopreservation enhances sperm cryosurvival. The aim of this study was to develop a CLC-treatment to optimize goat sperm cryopreservation. A total of 45 ejaculates coming from eleven adult Murciano-Granadina bucks were used and three experiments were conducted to determine: (1) the optimal CLC concentration to treat goat sperm; (2) the optimal time to treat the sperm (before or after seminal plasma removal); and (3) optimal freezing diluent (either of two Tris-citrate diluents containing 2% or 20% egg yolk and 4% glycerol or a skim milk diluent with 7% glycerol) to cryopreserve goat sperm. Goat sperm cryosurvival rates were greatest when they were treated with 1 mg CLC/120 × 10⁶ sperm prior to freezing. The benefit was also greatest if the sperm were treated with CLC after seminal plasma removal. Finally, CLC treatment improved sperm cryosurvival rates for sperm frozen in all three diluents, however, CLC treatment was most effective for sperm frozen in egg-yolk diluents. In conclusion, treating goat sperm, with CLC prior to cryopreservation, improved sperm cryosurvival rates. In addition, CLC treatment was effective for all freezing diluents tested, making this technology practical for the industry using current cryopreservation techniques. Nevertheless, additional studies should be conducted to determine how CLC might affect sperm functionality and fertilizing ability.