Freezing of fresh Barhi dates for quality preservation during frozen storage

Fresh harvested dates are perishable and there is a need for extending their shelf life while preserving their fresh like quality characteristics. This study evaluates three different freezing methods, namely cryogenic freezing (CF) using liquid nitrogen; individual quick freezing (IQF) and conventional slow freezing (CSF) in preserving the quality and stability of dates during frozen storage. Fresh dates were frozen utilizing the three methods. The produced frozen dates were frozen stored for nine months. The color values, textural parameters, and nutrition qualities were measured for fresh dates before freezing and for the frozen dates every three months during the frozen storage. The frozen dates’ color values were affected by the freezing method and the frozen storage period. There are substantial differences in the quality of the frozen fruits in favor of cryogenic freezing followed by individual quick freezing compared to the conventional slow freezing. The results revealed large disparity among the times of freezing of the three methods. The freezing time accounted to 10 min for CF, and around 80 min for IQF, and 1800 min for CSF method.     

The experimental study for efficacy and safety of pancreatic cryosurgery

Objective: This study was designed to basic information concerning the efficacy and safety of cryosurgery for pancreatic cancer. Fifteen healthy pigs were used to perform biochemical analysis and histological assessment. Methods: Following anesthesia and laparotomy, an argon–helium cryoprobe was inserted into the pancreas. The introduction of argon gas induced a rapid decrease in temperature to ?160 °C (Group I, 5 pigs) or ?110 °C (Group II, 5 pigs), respectively, resulting in ice-ball formation of 15–20 mm diameter after 5 min. Following freezing, helium gas was circulated in the probe tip to increase the temperature to 10–20 °C over 3 min to thaw. The freeze/thaw cycle was then repeated. Group III (3 pigs) had a cryoprobe inserted, but without freezing, and Group IV (2 pigs) included untreated or normal control animals. Levels of serum amylase (AMY), IL-6 and C-RP were measured prior to freezing and for 7 days following the procedure. All pigs were euthanized 7 days post-treatment and pancreases were examined histologically. Results: Neither hyperaemia, edema or hemorrhage were observed in the un-frozen parts of the pancreas. Histological assessment revealed a significant level of necrosis in the central and lateral regions of the tissue frozen within the ice-ball. All cellular ultrastructure was destroyed and only observable as a few of remaining nuclei with broken crests and degranulated mitochondria and rough endoplasmic reticulum. There was a significant increase of serum AMY levels for a brief period in both “deep frozen” and the “shallow frozen” groups. However, the AMY also increased in two pigs in the “normal control” group and one pig from the “inserted cryoprobe without freeze” control group. All experimental pigs appeared healthy until the sacrifice time. Conclusion: Cryosurgery is a safe and effective ablative procedure for pancreatic tissue resulting in minimal complications.     

Cryopreservation of human ovarian tissue: Comparison of rapid and conventional freezing

Cryopreservation, which is the most important procedure in ovarian tissue banking, can be divided into two methods: conventional freezing and rapid freezing. In previous study, the higher effectiveness of rapid freezing in comparison with the conventional freezing for human oocytes and embryos was shown. Data on comparison of these two methods for human ovarian tissue are limited. The aim of this study was to compare conventional freezing and rapid freezing for human ovarian tissue. Ovarian tissue fragments from 14 patients were transported to the laboratory within 22–25 h in a special, isolated transport box, which can maintain a stable temperature of between 5 and 8 °C for 36 h. Small pieces of ovarian tissue (1 × 1–1.5 × 0.7–1 mm) were randomly distributed into four groups: Group 1: control, fresh pieces immediately after receiving transport box, Groups 2 and 3: experimental pieces after rapid freezing/warming, and Group 4: experimental pieces after conventional freezing/thawing. All pieces were cultured in vitro for 14 days. The viability of the tissue by in vitro production of hormones and development of follicles after culture was evaluated. The level of estradiol 17-β and progesterone was measured using heterogeneous competitive magnetic separation immunoassay. For histological analysis, the number of viable and damaged follicles was counted. After culture of fresh tissue pieces (Group 1), rapidly frozen/warmed pieces (Groups 2 and 3), and conventionally frozen/thawed pieces (Group 4), the supernatants showed estradiol 17-β concentrations of 358, 275, 331, and 345 pg/ml, respectively, and progesterone concentrations of 3.02, 1.77, 1.99, and 2.01 ng/ml, respectively. It was detected that 96%, 36%, 39%, and 84% follicles for Groups 1, 2, 3, and 4, respectively, were normal. For cryopreservation of human ovarian tissue, conventional freezing is more promising than rapid freezing.     

The influence of cryopreservation and quick-freezing on the mechanical properties of tendons

In order to maintain their native properties, cryopreserved tendons are usually used in biomechanical research and in transplantation of allogenic tendon grafts. The use of different study protocols leads to controversy in literature and thus complicates the evaluation of the current literature. The aim of this study consisted in examining the influence of different freezing and thawing temperatures on the mechanical properties of tendons. 60 porcine tendons were frozen at either −80 °C or −20 °C for 7 days and thawed at room or body temperature for 240 or 30 min, respectively. A subgroup of ten tendons was quick-frozen with liquid nitrogen (−196 °C) for 2 s before cryopreservation. Biomechanical testing was performed with a material testing machine and included creep, cyclic and load-to-failure tests. The results showed that freezing leads to a reduced creep strain after constant loading and to an increased secant modulus. Freezing temperature of −80 °C increased the secant modulus and decreased the strain at maximum stress, whereas thawing at room temperature reduced the maximum stress, the strain at initial tendon failure and the Young’s Modulus. Quick-freezing led to increased creep strain after constant loading, increased strain at initial failure in the load-to-failure test, and decreased strain at maximum stress. When cryopreserving, tendons for scientific or medical reasons, freezing temperature of −20 °C and thawing temperature of 37.5 °C are recommended to maintain the native properties of tendons. A treatment with liquid nitrogen in the sterilization process of tendon allografts is inadvisable because it alters the tendon properties negatively.     

In situ cryopreservation of human embryonic stem cells in gas-permeable membrane culture cassettes for high post-thaw yield and good manufacturing practice

Current strategies for marine pollution monitoring are based on the integration of chemical and biological techniques. The sea urchin embryo-larval bioassays are among the biological methods most widely used worldwide. Cryopreservation of early embryos of sea urchins could provide a useful tool to overcome one of the main limitations of such bioassays, the availability of high quality biological material all year round. The present study aimed to determine the suitability of several permeant (dimethyl sulfoxide, Me₂SO; propylene glycol, PG; and ethylene glycol, EG) and non-permeant (trehalose, TRE; polyvinylpyrrolidone, PVP) cryoprotectant agents (CPAs) and their combination, for the cryopreservation of eggs and embryos of the sea urchin Paracentrotus lividus. On the basis of the CPAs toxicity, PG and EG, in combination with PVP, seem to be most suitable for the cryopreservation of P. lividus eggs and embryos. Several freezing procedures were also assayed. The most successful freezing regime consisted on cooling from 4 to −12 °C at 1 °C/min, holding for 2 min for seeding, cooling to −20 °C at 0.5 °C/min, and then cooling to −35 °C at 1 °C/min. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively.     

Alternative procedures for the cryopreservation of brown bear ejaculates depending on the flexibility of the “in cooling” period (5 °C)

The adaptability of cryopreservation protocols for brown bear spermatozoa collected under field conditions and frozen in a nearby laboratory (transported for a few hours) or shipped to a reference laboratory for sex sorting (transported for a few days) was evaluated. Forty-nine electroejaculates from 15 mature brown bears were extended to 100 × 10⁶ sperm/mL in a TES-Tris-Fructose based extender and cryopreserved (−20 °C/min to −100 °C and stored at −196 °C). After thawing, the quality of the seminal samples was assessed for total (TM), progressive (PM) motility and kinetic parameters – by CASA –, and viability (VIAB), viable and non-apoptotic status (YOPRO−), high membrane mitochondrial potential (MIT) and intact acrosomes (iACR) – by flow cytometry –. In Experiment 1, we assessed different storage times (0, 0.5, 1 – control –, 4–5, 7–8 and 11–12 h) at 5 °C from final dilution to freezing. After thawing, non-equilibrated samples (0 h) showed lower values of iACR, TM and PM. No significant differences were found for the different periods of equilibration tested. In Experiment 2, we evaluated three long-term storage times (24, 48 and 72 h) at 5 °C before freezing using storage for 1 h as control. The post-thawing quality of brown bear spermatozoa declined markedly after 48–72 h of pre-freezing. In conclusion, our findings suggest the possibility of extending the pre-freezing cooling period up to 24 h post-collection without freezing. This knowledge should enable the adaptation of the freezing protocols for when a special handling conditions are required such as the shipment of seminal samples to technological centers for the pre-freezing application of enhancer spermatic biotechnologies.     

Effects of preservation procedures of rumen inoculum on in vitro microbial diversity and fermentation

Sheep rumen contents were used as inoculum for an in vitro semi-continuous incubation system to study whether preservation method affects microbial fermentation pattern. Rumen fluid was filtered and either used immediately as inoculum (CTL) or dispensed into 110 mm × 16 mm tubes, that were stored refrigerated at 6 °C for 4 h (REF) or frozen at ?20 °C (FRZ), frozen in liquid N (FLN) or added with 0.04 glycerol and frozen in liquid N (FGL) for 48 h. Frozen inocula were thawed at 39 °C for 2 min before use (16 ml per bottle). Two 24 h incubations with four bottles per treatment were completed. The microbial utilisation of added glycerol after thawing in FGL increased total gas production (P<0.05) and 24 h volatile fatty acid (VFA) production (P<0.05), and also increased propionate and butyrate proportions at the expense of acetate. The other freezing inocula (i.e., FLN and FRZ) reduced the rate of gas production (as ml/g dry matter per hour), compared with CTL in the first 2 and 4 h of incubation (P<0.05), but this was compensated by increased fermentation at 8 and 12 h, respectively. Differences in gas production did not manifest a different VFA pattern at either 6 or 24 h incubation. Bacterial diversity was slightly affected by the preservation process, and the similarity index between untreated inocula and the 24 h incubated CTL samples was 0.690–0.724. Similarity between bacterial communities in FRZ and FLN with that in CTL after incubation was 0.678. The freezing preservation method of rumen inocula for subsequent in vitro gas production studies does not affect microbial fermentation pattern or bacterial biodiversity, provided that processing is rapid enough by using a high surface to volume ratio. Freezing in liquid N is more appropriate than at ?20 °C.     

Effects of the cooling mode on the structure and strength of porous scaffolds made of chitosan,alginate, and carboxymethyl cellulose by the freeze-gelation method

A freeze-gelation method was utilized to prepare porous scaffolds made of chitosan, alginate, and carboxymethyl cellulose because of their usefulness in tissue engineering applications. These polysaccharide solutions were cooled down to freezing using either a fast-cooling (FC) mode (>20 °C/min) or a slow-cooling (SC) mode (0.83 °C/min). Then the frozen polysaccharide solutions were immersed in their respective non-solvents to form porous scaffolds. Based on the SEM and optical microscope images of the scaffolds, the FC mode induced non-simultaneous nucleation and generated directional pore structures. In contrast, simultaneous nucleation and uniform and isotropic pore structures (mean pore size: 60–100 μm) were obtained by using the SC mode. Moreover, the tensile strength of the scaffolds prepared by the SC mode (about 60 N/g) was three times higher than that of scaffolds prepared by the FC mode (about 20 N/g). This study reveals that when using the freeze-gelation method, the cooling rate (mode) is a crucial factor which controls the pore structure and strength of porous scaffolds. Therefore, our results suggest that polysaccharide scaffolds with pore structures suitable for tissue engineering applications can be obtained via an appropriate cooling mode.     

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.     

DNA integrity of fresh and frozen canine epididymal spermatozoa

The aims of this study were to evaluate: (1) the effect of cryopreservation on DNA fragmentation of canine epididymal spermatozoa, and (2) the potential protective effect of melatonin on post-thaw sperm quality (motility, morphology, acrosomal and DNA integrity). Epididymal spermatozoa were collected after orchiectomy of ten dogs. Sperm samples were frozen in the presence or absence of melatonin (1 mM). DNA fragmentation index (percentage of spermatozoa with fragmented DNA) was similar in fresh samples (3.3 ± 3.6) and samples frozen with (4.2 ± 3.8) or without (3.6 ± 3.7) melatonin. Sperm motility was significantly (p < 0.0001) higher in fresh compared to frozen samples. The presence of melatonin in the freezing extender did not affect the sperm motility. Proportions of spermatozoa with normal morphology were similar in fresh and frozen samples, irrespective of the presence of melatonin in the extender. Acrosome integrity was significantly decreased (p < 0.01) by cryopreservation, and melatonin did not exert any beneficial effects. In conclusion, DNA fragmentation of canine epididymal spermatozoa was not affected by the freezing procedure, and the presence of melatonin did not preserve motility and acrosome integrity which were adversely affected by cryopreservation. The evaluation of DNA status of thawed gametes is particularly relevant for epididymal spermatozoa since these spermatozoa are usually stored and used in assisted reproductive techniques.     

Effect of cooling rate and equilibration time on pre-freeze and post-thaw survival of buck sperm

Survival of buck sperm is affected due to duration and temperature of stages of refrigerated or frozen storage. This study investigated interactive effect of cooling rates (moderate; MC and rapid cooling; RC); and equilibration times (0, 2, 4 and 8 h) on survival before freezing at 4 °C and post-thaw quality of buck sperm. Semen was collected (three Beetal bucks; replicates = 6), pooled and diluted with Tris-citrate extender. Pooled semen samples were subjected to either RC (−2.2 °C/min) or MC (−0.3 °C/min) from 37 °C to 4 °C in separate aliquots and further equilibrated at 4 °C for 8 h. Semen was frozen using standard procedure after completion of each equilibration period i.e. 0, 2, 4 and 8 h. Semen was evaluated for motility, viability, plasma membrane integrity (PMI) and normal apical ridge (NAR) before freezing and after thawing. The survival time (time for survival above threshold limit i.e. 60%) at 4 °C, of motility and PMI was observed 5 and 6 h respectively in RC group while >8 h in MC group. Rate of decline (slope) in motility and viability was higher (P < 0.05) in RC overtime during equilibration at 4 °C while PMI and NAR declined at equal rate in both cooling groups. Post-thaw motility and NAR were higher (P < 0.05) in MC when equilibrated for 2–8 h while viability and PMI of RC was observed equal to MC group. In conclusion, survival of buck sperm is higher when cooled with moderate rate. However, RC can maintain post-thaw sperm viability and PMI equal to MC when equilibrated for 2–8 h. The methods should be explored to maintain motility and NAR during rapid cooling of buck sperm.     

Effect of initial freezing temperature on the semen characteristics of frozen-thawed ram spermatozoa in a semi-arid tropical environment

Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including initial freezing temperature. The present study was conducted to observe the effect of initial freezing temperature on post-thawing motility of ram spermatozoa of native and crossbred rams maintained in a semi-arid tropical environment. Good quality semen obtained from native Malpura and crossbred Bharat Merino rams were pooled within breed and diluted at a rate of 1000 million spermatozoa per milliliter in TEST—yolk–glycerol extender. Diluted semen samples were loaded in 0.25 ml straws and cooled to −25, −75 or −125 °C freezing temperature at the rate of −25 °C/min under controlled conditions before plunging into liquid nitrogen for storage. The thawing of straws was performed at 50 °C in a water bath for 10 s and motility characteristics of the frozen-thawed spermatozoa were assessed by a computer-assisted spermatozoa analysis technique. Initial freezing temperature significantly affected the post-thawing motility of sperm in both the breeds. The post-thawing % motility and rapid motile spermatozoa were significantly higher at initial freezing temperature of −125 °C and lower at −25 or −75 °C. The percentage medium motile sperm were similar at all three initial freezing temperatures. The percentage of slow motile and linearity of sperm varied (P<0.01) between the different freezing temperatures. The curvilinear velocity, average path velocity and straight line velocity of spermatozoa were higher (P<0.01) at −125 °C than −25 or −75 °C. Although the lateral head displacement of spermatozoa did not vary significantly between the different initial freezing temperatures, the stroke frequency was significantly lower at −25 °C than −75 or −125 °C. Except for % linearity, the average path velocity and straight line velocity, other spermatozoa characteristics were not significantly different between breeds. The interaction between freezing temperature and breed was significant only for the % motility and linearity of the spermatozoa. The study indicates that initial freezing temperature has a significant effect on spermatozoa motility and velocity following post-thawing. The best motile spermatozoa following thawing were achieved at −125 °C freezing temperature.     

Characterization and evaluation of hydrophobically modified chitosan scaffolds: Towards design of enzyme immobilized flow-through electrodes

Chitosan scaffolds were fabricated by application of thermally induced phase separation from aqueous solutions of unmodified chitosan and hydrophobically modified chitosan polymer. The final pore structure, in terms of diameter and geometry, were correlated to freezing temperature and freezing time for both the unmodified and hydrophobically modified chitosan polymer. Results showed that the resulting pore structure is strongly dependent upon the freezing temperature and less dependant upon the freezing time. For scaffolds produced from unmodified chitosan, the pore size decreased as expected with decreasing freezing temperature from ?5 °C to ?10 °C. However, an inconsistency in this trend was observed as the freezing temperature was decreased to ?20 °C. Combined analysis of pore size distribution and average pore diameter suggested that the freezing process was mainly mass transfer dominated at ?5 °C and ?10 °C, but principally heat transfer dominated at ?20 °C. In comparison, the scaffolds produced from hydrophobically modified chitosan (butyl-chitosan) followed the expected trend of decreasing mean pore diameter with decreased freezing temperatures throughout the entire temperature range. The scaffolds produced from the unmodified chitosan were more stable and rigid, and possessed average pore diameters that were generally smaller than those fabricated from the hydrophobically modified chitosan. The generally larger pores in the butyl-modified chitosan scaffolds might be explained by increased phase separation rates due to the introduced hydrophobicity of the chitosan polymer. Among the scaffolds fabricated from the butyl-modified chitosan, those produced at ?20 °C yielded the most uniform pore structure, the smallest average pore diameters, and the least temporal broadening of pore size distribution.     

Assessment of frozen larvae of Callosobruchus maculatus as hosts for rearing Pteromalus cerealellae (Ashmead) (Hymenoptera: Pteromalidae)

The suitability of frozen host larvae for rearing Pteromalus cerealellae (Ashmead) (Hymenoptera: Pteromalidae), an ectoparasitoid of Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae) and other stored-product insects was investigated. The reproductive potential (number and sex ratio of progeny) of female P. cerealellae was compared on live (fresh) C. maculatus larvae (concealed within cowpea seeds) versus frozen larvae (obtained by freezing infested cowpea seeds at ?20 °C for 48 h) which were subsequently thawed and held at ambient conditions (～25 ± 1 °C, 50 ± 5% RH) for 4, 24, 48, 72, 96, and 120 h before exposure to female parasitoids. No significant differences were recorded in the numbers and sex ratios of the progeny produced by female P. cerealellae on live larvae compared to frozen host larvae that were thawed and held at ambient conditions for up to 96 h, suggesting that live and frozen larvae of C. maculatus are equally suitable for rearing P. cerealellae. However, the data showed that progeny production on frozen hosts gradually declined with thawing duration and was significantly reduced at the thawing duration of 120 h. When live and frozen host larvae were simultaneously presented together to female P. cerealellae at different exposure periods, relatively greater progeny production was recorded on live hosts than on frozen hosts at 12, 24, and 48 h of exposure. This may suggest preference of female P. cerealellae for live versus frozen host larvae. These results are discussed in relation to the life history strategy and host location behavior of P. cerealellae, and may have practical implications in the development of efficient mass rearing systems for the parasitoid.     

Freeze tolerance and accumulation of cryoprotectants in the enchytraeid Enchytraeus albidus (Oligochaeta) from Greenland and Europe

The freeze tolerance and accumulation of cryoprotectants was investigated in three geographically different populations of the enchytraeid Enchytraeus albidus (Oligochaeta). E. albidus is widely distributed from the high Arctic to temperate Western Europe. Our results show that E. albidus is freeze tolerant, with freeze tolerance varying extensively between Greenlandic and European populations. Two populations from sub Arctic (Nuuk) and high Arctic Greenland (Zackenberg) survived freezing at −15 °C, whereas only 30% of a German population survived this temperature. When frozen, E. albidus responded by catabolising glycogen to glucose, which likely acted as a cryoprotectant. The average glucose concentrations were similar in the three populations when worms were frozen at −2 °C, approximately 50 μg glucose mg⁻¹ tissue dry weight (DW). At −14 °C the glucose concentrations increased to between 110 and 170 μg mg⁻¹ DW in worms from Greenland. The average glycogen content of worms from Zackenberg and Nuuk were about 300 μg mg⁻¹ DW, but only 230 μg mg⁻¹ DW in worms from Germany showing that not all glycogen was catabolised during the experiment. Nuclear magnetic resonance spectrometry (NMR) was used to screen for other putative cryoprotectants. Proline, glutamine and alanine were up regulated in frozen worms at −2 °C but only in relatively small concentrations suggesting that they were of little significance for freeze survival. The present study confirms earlier reports that freeze tolerant enchytraeids, like other freeze tolerant oligochaete earthworms, accumulate high concentrations of glucose as a primary cryoprotectant.     

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).     

24-gauge ultrafine cryoprobe with diameter of 550 μm and its cooling performance

This paper describes the development of a novel cryoprobe with the same size as a 24-gauge injection needle and the evaluation of its cooling performance. This ultrafine cryoprobe was designed to reduce the invasiveness and extend application areas of cryosurgery. The ultrafine cryoprobe has a double-tube structure and consists of two stainless steel microtubes. The outer diameter of the cryoprobe is 550 μm, and the inner tube has a 70-μm inner diameter to depressurize the high-pressure refrigerant. By solving the bioheat transfer equation and considering freezing phenomena, the relationship between the size of the frozen region and the heat transfer coefficient of the refrigerant flow in an ultrafine cryoprobe was derived analytically. The results showed that the size of the frozen region is strongly affected by the heat transfer coefficient. A high heat transfer coefficient such as that of phase change heat transfer is required to generate a frozen region of sufficient size. In the experiment, trifluoromethane (HFC-23) was used as the refrigerant, and the cooling effects of the gas and liquid phase states at the inlet were evaluated. When the ultrafine cryoprobe was cooled using a liquid refrigerant, the surface temperature was approximately −50 °C, and the temperature distribution on the surface was uniform for a thermally insulated condition. However, for the case with vaporized refrigerant, the temperature distribution was not uniform. Therefore, it was concluded that the cooling mechanism using liquid refrigerant was suitable for ultrafine cryoprobes. Furthermore, to simulate cryosurgery, a cooling experiment using hydrogel was conducted. The results showed that the surface temperature of the ultrafine cryoprobe reached −35 °C and formed a frozen region with a radius of 4 mm in 4 min. These results indicate that the ultrafine cryoprobe can be applied in actual cryosurgeries for small affected areas.     

Comparison of the effects of glutamine and an amino acid solution on post-thawed ram sperm parameters,lipid peroxidation and anti-oxidant activities

Ram semen contains sufficient quantities of superoxide dismutase (SOD) and much lower concentrations of glutathione peroxidase (GSH-PX) and catalase (CAT) to prevent oxidative damage. The anti-oxidant capacity of the sperm cell is limited, due to a small cytoplasmic component, which contains these anti-oxidants to scavenge the oxidants. However, the concentration of these anti-oxidants may decrease considerably by the dilution of the semen. The aim of the present work was to study the effect of two anti-oxidants, namely, glutamine and an amino acid solution (BME) in a Tris-based extender on ram sperm parameters, lipid peroxidation and anti-oxidant capacity after the cryopreservation/thawing process. Ejaculates collected from 4 Akkaraman rams were evaluated and pooled at 37 °C. Semen samples which were diluted with the tris-based extender containing glutamine (2.5 or 5 mM), BME (13 or 26%), and no anti-oxidants (control) were cooled to 5 °C and frozen in 0.25-ml French straws and stored in liquid nitrogen. Frozen straws were thawed individually at 37 °C for 20 s in a water bath for evaluation. The freezing extender supplemented with 5 mM glutamine led to higher motility rate (68.0 ± 4.4%) and hypo-osmotic swelling test (HOST) (64.1 ± 5.5%), when compared to glutamine (2.5 mM) and BME (13 and 26%) (P < 0.05). No significant differences were observed regarding sperm motility and HOST, following the supplementation of the freezing extender with glutamine 2.5 mM and BME (13 and 26%) after thawing. CAT activity remained significantly higher following the addition of glutamine 5 mM (6.4 ± 0.9 kU/g protein), compared to the other treatments (P < 0.01). The anti-oxidants at different levels were not effective in the elimination of malondialdehyde (MDA) formation and maintenance of SOD activities, when compared to the control (P < 0.05). Findings showed that glutamine (5 mM) supplementation in semen extenders, was of greater benefit to frozen–thawed ram sperm. Future efforts are needed to find the appropriate anti-oxidants and their effective concentrations to improve post-thaw sperm parameters (e.g. motility, membrane integrity, fertility) and anti-oxidant activities when frozen–thawed ram sperm is used.     

Overwintering temperatures affect freezing temperatures of turions of aquatic plants

The effect of different overwintering temperatures (2.5 ± 1 °C in a refrigerator or outdoor natural overwintering on wet topsoil with weak frosts) on the freezing temperature and survival rate of turions of 10 aquatic plant species with different ecological traits (free-floating habit or bottom rooting) was studied using mini thermocouples. Dormant, non-hardened turions of 9 species exhibited freezing within a narrow temperature range of ?7.0 to ?10.2 °C, while Hydrocharis morsus-ranae froze at ?3.6 °C. The survival rate of the turions after the measurements was, however, very low (0–38%). In several species, the freezing temperature of turions at the beginning of germination was not significantly different (at p < 0.05) from the dormant ones. The mean freezing temperature of outdoor hardened turions of 6 species was within a very narrow range of ?2.8 to ?3.3 °C and was thus significantly higher by 4–7 °C (p < 0.0002) than that for the non-hardened turions. It is assumed that the freezing temperatures indicate freezing of the extracellular water. The hardened turions of all 7 species were able to survive mild winter frosts under the topsoil conditions at a rate of 76–100%. These characteristics suggest that the turions of aquatic species can be hardened by weak frosts and that their frost hardiness is based on the shift from frost avoidance in non-hardened turions to frost tolerance.     

Determination of heat transfer coefficients in plastic French straws plunged in liquid nitrogen

The knowledge of the thermodynamic process during the cooling of reproductive biological systems is important to assess and optimize the cryopreservation procedures. The time–temperature curve of a sample immersed in liquid nitrogen enables the calculation of cooling rates and helps to determine whether it is vitrified or undergoes phase change transition. When dealing with cryogenic liquids, the temperature difference between the solid and the sample is high enough to cause boiling of the liquid, and the sample can undergo different regimes such as film and/or nucleate pool boiling.In the present work, the surface heat transfer coefficients (h) for plastic French straws plunged in liquid nitrogen were determined using the measurement of time–temperature curves. When straws filled with ice were used the cooling curve showed an abrupt slope change which was attributed to the transition of film into nucleate pool boiling regime. The h value that fitted each stage of the cooling process was calculated using a numerical finite element program that solves the heat transfer partial differential equation under transient conditions. In the cooling process corresponding to film boiling regime, the h that best fitted experimental results was h = 148.12 ± 5.4 W/m² K and for nucleate-boiling h = 1355 ± 51 W/m² K. These values were further validated by predicting the time–temperature curve for French straws filled with a biological fluid system (bovine semen-extender) which undergoes freezing. Good agreement was obtained between the experimental and predicted temperature profiles, further confirming the accuracy of the h values previously determined for the ice-filled straw. These coefficients were corroborated using literature correlations.The determination of the boiling regimes that govern the cooling process when plunging straws in liquid nitrogen constitutes an important issue when trying to optimize cryopreservation procedures. Furthermore, this information can lead to improvements in the design of cooling devices in the cryobiology field.