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.     

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.     

Freezing of boar semen can be simplified by handling a specific portion of the ejaculate with a shorter procedure and MiniFlatPack packaging

Low sperm survival post-thaw and time-consuming procedures for conventional freezing (CF) hamper the commercial application of cryopreserved boar semen. We had previously proven that boar spermatozoa in the first 10 mL of the sperm-rich fraction, SRF (the so-called P1, the sperm-peak portion of the ejaculate) sustain best handling in vitro, since they probably bathe in an aliquot of seminal plasma (SP) with specific composition. Here, we performed three experiments to determine: Exp I: the concentration of bicarbonate among portions of the ejaculate; Exp II: the effects of bicarbonate doses on sperm motility and; Exp III: the outcome of a faster, simpler freezing method (SF), handling P1-spermatozoa packed in MiniFlatPacks? (MFP) vs. CF and vs. SRF-spermatozoa (2 × 2 factorial design). The bicarbonate content in SP was, among portions/fractions of the ejaculate, lowest in P1 (13.71 mM/L, P < 0.0001, Exp I). Boar spermatozoa require bicarbonate in the extender (to the levels present in P1) to maintain acceptable motility over a 120-h period at 16–17 °C (Exp II). Sperm freezing was dramatically shortened (from 8 to 3.5 h) by the SF-procedure. P1- and SRF-spermatozoa survived equally both CF- and SF-freezing (% total motility 30 min PT; P1-CF: 65.2 ± 5.4% and P1-SF: 68.9 ± 2.4%; SRF-CF: 64.4 ± 2.7%; SRF-SF: 55.8 ± 3.1%, ns). Interestingly, in contrast to SRF, there were no significant variations in 30-min PT-survival among either ejaculates or boars when the P1 was frozen, independent of the handling method (CF or SF). In conclusion, such a faster freezing protocol of semen packed in MFP could be advantageously applied to P1-spermatozoa (P1-SF), while the rest of the ejaculated spermatozoa could still be used for production of conventional artificial insemination (AI) doses, thus allowing for a maintained routine management of commercially relevant stud boars.     

Motility and fertility of cryopreserved semen in Persian sturgeon,Acipenser persicus,stored for 30–60 min after thawing

We investigated the effect of storage times of frozen–thawed Persian sturgeon (Acipenser persicus) semen on the duration of sperm motility, percentage of motile sperm, and fertilization and hatching rates of fresh sperm and sperm stored for 0, 30, and 60 min at 4 °C post-thawing. Frozen thawed semen analyzed immediately after thawing had similar quality characteristics as fresh semen. For cryopreserved semen stored for 30 min after thawing the characteristics did not differ to fresh semen and cryopreserved semen. For cryopreserved semen stored for 60 min a significant decline in the parameters was observed. Fertilization and hatching rates were not affected by storage times of maximally 30 min of storage.     

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.     

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.     

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.     

Changes in carotenoid and chlorophyll content of black tomatoes (Lycopersicone sculentum L.) during storage at various temperatures

Black tomatoes have a unique color and higher lycopene content than typical red tomatoes. Here, black tomatoes were investigated how maturation stage and storage temperature affected carotenoid and chlorophyll accumulation. Immature fruits were firmer than mature fruits, but failed to develop their distinctive color and contained less lycopene when stored at 8 °C. Hunter a^* values of black tomatoes increased with storage temperature and duration; storage of immature fruits at high temperature favored lycopene accumulation. Chlorophyll levels of black tomatoes declined during storage, but differences between mature and immature tomatoes stored at 12 °C were minimal. β-Carotene levels of black tomatoes increased during early storage, but rapidly declined beginning 13 d post-harvest. The highest lycopene and chlorophyll levels were observed in mature black tomatoes stored at 12 °C for 13 d; these conditions also yielded the best quality fruit. Thus, the unique pigmentation properties of black tomatoes can be precisely controlled by standardizing storage conditions.     

Effect of storage of domestic cat (Felis catus) epididymides at 5 °C on sperm quality and cryopreservation

Postmortem sperm recovery from the epididymides may constitute a powerful tool for the conservation of valuable genetic material. The domestic cat (Felis catus) is a good model for wild felids and, using this model, we have explored the effect of epididymides storage time on sperm motility and percentage of intact acrosomes upon sperm recovery and after cryopreservation. We also examined the effect of time of sperm equilibration with glycerol before freezing on sperm motility and the percentage of intact acrosomes. Motility varied between sperm recovered from epididymides that were stored for different times. Significant differences were seen in the sperm motility index (SMI) before freezing (55.91 ± 2.02, 48.21 ± 1.47, and 43.03 ± 1.32) and after thawing (51.81 ± 3.02, 41.90 ± 2.14, and 42.35 ± 1.95) of sperm recovered from epididymides stored for 0, 48, or 72 h, respectively. The percentage of intact acrosomes did not vary significantly with storage time (average 60.33 ± 1.38% before and 52.50 ± 1.91% after freezing, respectively). The percentage of normal sperm after different storage times did not differ (average 19.22 ± 1.25% normal sperm after recovery). When epididymides were stored for 72 h, time of sperm equilibration with glycerol (30 vs. 120 min) resulted in significant differences in both motility (SMI = 39.17 ± 2.76 and 45.00 ± 2.65, respectively) and the percentage of intact acrosomes (45.76 ± 4.91% and 60.67 ± 3.64%, respectively) after thawing. In conclusion, best results are achieved when sperm are recovered from epididymides within 24 h of cool storage and when they are equilibrated with glycerol during 120 min before freezing. The current results should be useful in the further development of techniques for the rescue and cryostorage of epididymal spermatozoa of endangered felids.     

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.     

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.     

Effects of simulated warming on soil ammonia-oxidizing bacteria and archaea communities in an alpine forest of western Sichuan,China

Ongoing climate change, characterized by winter warming, snow cover decline and extreme weather events, is changing terrestrial ecosystem processes in high altitude and latitude regions. Winter soil processes could be particularly sensitive to climate change. In fact, winter warming and snow cover decline are interdependent in cold biomes, and have a synergistic effect on soil processes. Soil microorganisms not only play crucial roles in material cycling and energy flow, but also act as sensitive bio-indicators of climate change. However, little information is available on the effect of winter warming on forest soil ammonia-oxidizing bacteria (AOB) and archaea (AOA). The alpine and subalpine forest ecosystems on the eastern Tibet Plateau have important roles in conserving soil, holding water, and maintaining biodiversity. To understand the changes in AOB and AOA communities under climate change scenarios, an altitudinal gradient experiment in combination with soil column transplanting was conducted at the Long-term Research Station of Alpine Forest Ecosystems, which is situated in the Bipeng Valley of Lixian County, Sichuan, China. Thirty intact soil columns under an alpine forest at an altitude of 3582 m were transplanted and incubated at 3298 m and 3023 m forest sites, respectively. Compared with the 3582 m, we expected air temperature increases of 2 °C and 4 °C at the 3298 m and 3023 m, respectively. However, the temperatures in the soil organic layer (OL) and mineral soil layer (ML) increased by 0.27 °C and 0.13 °C, respectively, at 3023 m and ? 0.36 °C and ? 0.35 °C at 3298 m. Based on a previous study and with simultaneous monitoring of soil temperature, the abundances of AOB and AOA communities in both the OL and ML were measured by qPCR in December 2010 (i.e., the onset of the frozen soil period) and March 2011 (i.e., the late frozen soil period). The soil columns incubated at 3023 m had relatively higher AOB abundances and lower AOA/AOB ratios than those at 3298 m, while higher AOA abundances and AOA/AOB ratios were observed at 3298 m. The abundance of the microbial community at the late frozen period was higher than that at the onset of frozen soil, and the changes in microbial community abundance at the late frozen period were more substantial. Furthermore, the nitrate nitrogen (N) concentrations in both the OL and ML were significantly higher than ammonia N concentrations, implying that soil nitrate N is the primary component of the inorganic N pool in the alpine forest ecosystem. Additionally, the responses of AOA and AOB in the soil OL to soil column transplanting were more sensitive than the responses of those in ML. In conclusion, climate warming alters the abundance of the ammonia-oxidizing microbial community in the alpine forest ecosystem, which, in turn, might affect N cycling.     

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.     

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.     

Photo-controlled release of fipronil from a coumarin triggered precursor

Developing efficient controlled release system of insecticide can facilitate the better use of insecticide. We described here a first example of photo-controlled release of an insecticide by linking fipronil with photoresponsive coumarin covalently. The generated coumarin-fipronil (CF) precursor could undergo cleavage to release free fipronil in the presence of blue light (420 nm) or sunlight. Photophysical studies of CF showed that it exhibited strong fluorescence properties. The CF had no obvious activity against mosquito larvae under dark, but it can be activated by light inside the mosquito larvae. The released Fip from CF by blue light irradiation in vitro retained its activity to armyworm (Mythimna separate) with LC₅₀ value of 24.64 μmol L^?1. This photocaged molecule provided an alternative delivery method for fipronil.     

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.     

Amphipathic polymer-mediated uptake of trehalose for dimethyl sulfoxide-free human cell cryopreservation

For stem cell therapy to become a routine reality, one of the major challenges to overcome is their storage and transportation. Currently this is achieved by cryopreserving cells utilising the cryoprotectant dimethyl sulfoxide (Me₂SO). Me₂SO is toxic to cells, leads to loss of cell functionality, and can produce severe side effects in patients. Potentially, cells could be frozen using the cryoprotectant trehalose if it could be delivered into the cells at a sufficient concentration. The novel amphipathic membrane permeabilising agent PP-50 has previously been shown to enhance trehalose uptake by erythrocytes, resulting in increased cryosurvival. Here, this work was extended to the nucleated human cell line SAOS-2. Using the optimum PP-50 concentration and media osmolarity, cell viability post-thaw was 60 ± 2%. In addition, the number of metabolically active cells 24 h post-thaw, normalised to that before freezing, was found to be between 103 ± 4% and 91 ± 5%. This was found to be comparable to cells frozen using Me₂SO. Although reduced (by 22 ± 2%, p = 0.09), the doubling time was found not to be statistically different to the non-frozen control. This was in contrast to cells frozen using Me₂SO, where the doubling time was significantly reduced (by 41 ± 4%, p = 0.004). PP-50 mediated trehalose delivery into cells could represent an alternative cryopreservation protocol, suitable for research and therapeutic applications.     

Variation the oviposition behavior by the stingless bee,Heterotrigona itama (Hymenoptera,Apidae, Meliponini)

A study to determine the variation of ovipositioning behavior of stingless bees, Heterotrigona itama (Cockerell, 1918) was conducted on three colonies on June 2015. A digital single-lens reflex (DSLR) camera with a macro lens attached was used to record every movement of H. itama in its colonies for 20 min hour between 0800 h and 2000 h for seven days and seven month. The daily egg laying rate and time for laying eggs in colony-B and colony-C were significantly (P ? 0.05) higher than the colony-A. However, time to close the brood was not significantly different among colonies. The fastest egg oviposition time was 4 s by the colony-B and the slowest was 6 s by the colony-A. In addition there are no significant trends on brood produced per day, laying time of eggs, and the closing time of the brood after the oviposition process from June to December 2016. This result is useful for understanding the behavior of egg laying process by the queen bees and necessary to deal with problems of management and reproduction in the near future.     

Effect of the holding time at 15 °C prior to cryopreservation,the thawing rate and the post-thaw incubation temperature on the boar sperm quality after cryopreservation

The aim of the present study was to evaluate the effect of the holding time at 15 °C prior to cryopreservation (2, 4 and 8 h), thawing rate (37 °C for 20 s or 70 °C for 8 s) and post-thaw incubation temperature (15 °C or 37 °C) on the post-thaw boar sperm quality. These are important time periods in the freezing–thawing process which have been less studied. Sperm-rich ejaculate fractions from three healthy boars were collected once a week for five consecutive weeks and were cryopreserved with the lactose-egg yolk extender (LEY). Sperm quality was determined by assessing the motility, the acrosome status, and the sperm plasma membrane integrity at 30, 150 and 240 min of incubation. The results show that with the holding time at 15 °C prior to cryopreservation there was not a clear effect until at least 24 h of holding time. The thawing rate and the post-thaw incubation temperature, however, had a marked effect on sperm quality. When the samples were thawed at 70 °C for 8 s, the sperm viability, motility and some kinetic variables (VCL, VSL, VAP and ALH) were greater than with results observed when the samples were thawed at 37 °C for 20 s. In addition after thawing the sperm samples incubated at 15 °C had a sustained sperm quality for longer, up to 4 h post-thawing.     

Modélisation des flux cérébraux par une analogie électrique : validation par vélocimétrie IRM

The cine Phase-Contrast Magnetic Resonance (PCMR) sequence is the only noninvasive technique for the study of cerebrospinal fluid (CSF) oscillations. It can provide CSF and blood flow measurements throughout the cardiac cycle. To study cerebral hydro-hemodynamic, models have been developed; nevertheless the majority of these models did not take into account the CSF oscillations. The objective of this study was to establish reference values for cerebral hydro-hemodynamic and propose a new electrical model of the brain dynamics.Material and methodsCSF and blood flows were measured in 19 control subjects by PCMR imaging. Dynamic flow images were analyzed on dedicated software to reconstruct the flow curves during the cardiac cycle. An electrical analogue was realized. The inputs of the model were fed by PCMR arterial and venous flows to simulate CSF oscillations. The simulated CSF oscillations were compared to the measured CSF oscillations to validate the model.ResultsThe key parameters of the CSF and blood flow curves were obtained, e.g. total cerebral blood flow was 688 ± 115 mL/min, ventricular CSF oscillatory volume was 0.05 ± 0.02 mL/cardiac cycle, and the subarachnoid CSF oscillatory volume was 0.55 ± 0.15 mL/cardiac cycle. A close agreement was found between measured and simulated cerebral CSF oscillations.ConclusionThis study established the main values characterizing cerebral hydrodynamics in a control population. It provided a better understanding of the mechanisms of intracranial volumes regulation during the cardiac cycle. Our results are now used in clinical practice and the model proposed is effective to study cerebral hydro-hemodynamic.