Probe ultrasonification of egg yolk plasma forms low-density lipoprotein nanoparticles that efficiently protect canine semen during cryofreezing.

Around the world, many couples have turned to in vitro fertilization as a viable solution to fertility issues. Low-density lipoprotein (LDL) is a protein best known for transporting fat molecules throughout the body, but it has also been shown to protect sperm cells during cryopreservation due to its micellar structure. In the present study, we aimed to evaluate different protocols for the preparation of nanoparticles from egg yolk plasma (EYP) containing LDL to improve the viability of cryopreserved canine semen. EYP was subjected to three distinct treatments: ultrasonification in an ultrasound bath at 40 kHz for 30 min (LDL-B); ultrasonification via an ultrasound probe at 50% amplitude for 30 min (LDL-P); and high-pressure homogenization at 10,000 PSI for six cycles (LDL-H). Sperm quality was assessed after thawing using computer-assisted sperm analysis and flow cytometry. The results revealed that compared to the EYP control, the LDL-P formulation presented significantly higher efficiency (p < 0.05) in maintaining total and progressive sperm motility, sperm membrane integrity and fluidity, and levels of intracellular reactive oxygen species. The LDL-P nanoparticles had an average size of approximately 250 nm, a PDI value of 0.3, and -1.15 mV of zeta potential, which are very important because it is an indicator of the stability of a colloidal dispersion. Therefore, we conclude that ultrasonication of EYP using a probe is an efficient method for the preparation of LDL nanoparticles that would enhance the cryoprotection of semen during freezing.

Effect of cryoprotectants on rat kidney decellularization by freeze-thaw process.

To overcome the shortage of organ donors and morbidity and mortality caused by lifetime immunosuppression, development of a transplantable graft to permanently replace the organ function is required. This study is focused on the effects of a freeze-thaw process and cryoprotectants on the ultrastructure and composition of decellularization scaffolds. Results showed that cryoprotectants and freezing temperatures had significant effects on the decellularization scaffold. The vascular network integrity at -20 °C was better than that at -80 °C. For low-concentration cryoprotectants, 10% dimethyl sulfoxide and 5% trehalose could achieve a better balance between preserving the vascular tree and decellularization. For high-concentration cryoprotectants (vitrification solutions VS55 and VS83), the vascular network integrity was best because of the absence of freezing damage and ice-induced disruption of cells, but the decellularization effect was poor because the cells remained in the scaffold. Collagen, elastic fiber, protein, and mechanical properties of the scaffold could be retained after decellularization using the freeze-thaw method. Further studies and further optimization of the freeze-thaw decellularization protocol are necessary for clinical applications.

Effect of cryoprotectant on the motility, viability, fertilization, and DNA integrity of naleh fish Barbonymus sp. (Cyprinidae) sperm / Efeito do crioprotetor na motilidade, viabilidade, fertilização e integridade do DNA no sêmen de peixes naleh Barbonymus sp. (Cyprinidae)

Naleh fish Barbonymus sp. is a commercial freshwater fish, which is indigenous to Aceh, Indonesia. The population of this species has declined over the years as a result of habitat perturbations and overfishing. Hence, the crucial need to develop a cryopreservation method to support breeding programs. This involved the use of a cryoprotectant as an important component. The objective of this study, therefore, was to explore the best cryoprotectant for naleh fish spermatozoa, and a total of five types were tested. These include the DMSO, Methanol, Ethanol, Glycerol, and Ethylene Glycol at a similar concentration of 10%, which were individually combined with 15% egg yolk, and every treatment was performed in three replications. Conversely, Ringer's solution was adopted as an extender, and the sperm was cryopreserved in liquid nitrogen for 15 days. The results showed significant influence on sperm motility and viability, as well as egg fertility of naleh fish (P <0.05), although the DMSO provided the best outcome, compared to others at 47.17%, 50.13%, and 45.67%, respectively. Furthermore, DNA fragmentation had not occurred in the fresh and cryopreserved sperm samples, indicating the protective effect of tested cryoprotectants. It is concluded that the 10% DMSO and 15% egg yolk is the best cryoprotectant for naleh fish spermatozoa.(AU)

O peixe naleh Barbonymus sp. é um peixe comercial de água doce, originário de Aceh, Indonésia. Durante vários anos, as perturbações provocadas no seu habitat e a pesca predatória determinaram o declínio da sua população, cuja preservação deve apoiar-se em um programa de reprodução controlada, com o emprego de espermatozoides criopreservados. O presente trabalho realizou um estudo comparativo de cinco crioprotetores: dimetilsultóxido, metanol, etanol, glicerol e etileno glicol. Todos os crioprotetores foram testados na concentração de 10%, combinados a 15% de gema de ovo. Cada tratamento foi efetuado em triplicatas. A solução de ringer foi utilizada como extensor e o esperma foi criopreservado em nitrogênio líquido por 15 dias. Os resultados obtidos revelaram a existência de influência significante (P<0,05) na viabilidade e motilidade espermática bem como na fertilidade dos ovos do peixe naleh, em que o dimetilsulfóxido apresentou o melhor resultado com os valores de 47,17%, 50,13% e 45,67%, respectivamente. Por outro lado, a fragmentação do DNA não ocorreu nas amostras de esperma fresco e criopreservado, indicando o efeito protetor dos crioprotetores testados. A conclusão obtida foi que o dimetilsulfóxido e 15% de gema de ovo foram o melhor crioprotetor para os espermatozoides do peixe naleh.(AU)

Energy balance and metabolic changes in an overwintering wolf spider, Schizocosa stridulans.

Winter provides many challenges for terrestrial arthropods, including low temperatures and decreased food availability. Most arthropods are dormant in the winter and resume activity when conditions are favorable, but a select few species remain active during winter. Winter activity is thought to provide a head start on spring growth and reproduction, but few studies have explicitly tested this idea or investigated tradeoffs associated with winter activity. Here, we detail biochemical changes in overwintering winter-active wolf spiders, Schizocosa stridulans, to test the hypothesis that winter activity promotes growth and energy balance. We also quantified levels of putative cryoprotectants throughout winter to test the prediction that winter activity is incompatible with biochemical adaptations for coping with extreme cold. Body mass of juveniles increased 3.5-fold across winter, providing empirical evidence that winter activity promotes growth and therefore advancement of spring reproduction. While spiders maintained protein content throughout most of the winter, lipid content decreased steadily, suggesting either a lack of available prey to maintain lipids, or more likely, an allometric shift in body composition as spiders grew larger. Carbohydrate content showed no clear seasonal trend but also tended to be higher at the beginning of the winter. Finally, we tested the hypothesis that winter activity is incompatible with cryoprotectant accumulation. However, we observed accumulation of glycerol, myo-inositol, and several other cryoprotectants, although levels were lower than those typically observed in overwintering arthropods. Together, our results indicate that winter-active wolf spiders grow during the winter, and while cryoprotectant accumulation was observed in the winter, the modest levels relative to other species could make them susceptible to extreme winter events.

Human oocytes and zygotes are ready for ultra-fast vitrification after 2 minutes of exposure to standard CPA solutions.

Vitrification of human oocytes and embryos in different stages of development is a key element of daily clinical practice of in vitro fertilization treatments. Despite the cooling and warming of the cells is ultra-fast, the procedure as a whole is time consuming. Most of the duration is employed in a long (8-15 minutes), gradual or direct exposure to a non-vitrifying cryoprotectant solution, which is followed by a short exposure to a more concentrated vitrifying solution. A reduction in the duration of the protocols is desirable to improve the workflow in the IVF setting and reduce the time of exposure to suboptimal temperature and osmolarity, as well as potentially toxic cryoprotectants. In this work it is shown that this reduction is feasible. In silico (MatLab program using two-parameter permeability model) and in vitro observations of the oocytes' osmotic behaviour indicate that the dehydration upon exposure to standard cryoprotectant solutions occurs very fast: the point of minimum volume of the shrink-swell curve is reached within 60 seconds. At that point, intracellular water ejection is complete, which coupled with the permeation of low molecular weight cryoprotectants results in similar intracellular and extracellular solute concentrations. This shows that prolonging the exposure to the cryoprotectant solutions does not improve the cytosolic glass forming tendency and could be avoided. To test this finding, human oocytes and zygotes that were donated for research were subjected to a shortened, dehydration-based protocol, consisting of two consecutive exposures of one-minute to two standard cryoprotectant solutions, containing ethylene glycol, dimethyl sulfoxide and sucrose. At the end of this two-minute dehydration protocol, the critical intracellular solute concentration necessary for successful vitrification was attained, confirmed by the post-warming survival and ability to resume cytokinesis of the cells. Further studies of the developmental competency of oocytes and embryos would be necessary to determine the suitability of this specific dehydration protocol for clinical practice, but based on our results, short times of exposure to increasingly hypertonic solutions could be a more time-efficient strategy to prepare human oocytes and embryos for vitrification.

Detection and Cryoprotective Activity of Dehydrin Proteins from Rice Bran and Soybean Whey.

Dehydrin proteins, group2 LEA proteins in rice bran and soybean whey were analyzed by two-dimensional (2D) electrophoresis, and the cryoprotective activity on freeze/thaw inactivation of lactate dehydrogenase was examined as a criterion of its function. Dehydrins in rice bran were detected by immunoblotting using antibody raised against a conserved lysine-rich motif sequence. In the water-soluble fraction of rice bran, 10 spots of 44 kDa and 23 kDa dehydrin-like polypeptides were detected on the immunoblotted membrane. Isoelectric points of the polypeptides were between 6.6 and 7.4. The 23 kDa dehydrin polypeptide was partially purified by ammonium sulfate fractionation and ion exchange column chromatography. CP50 value, protein amount necessary to keep 50% of enzyme activity, of the 23 kDa dehydrin was 0.78 µM (15.6 µg/mL), slightly lower than that of bovine serum albumin. Heat-soluble soybean whey proteins were analyzed by SDS-PAGE and 2D-electrophoresis. Dehydrin appeared to be the most abundant protein in the fraction. CP50 value for heat-soluble whey protein was estimated to be 15.8 µg/mL, while that of total whey was 355 µg/mL. The result indicated that simple heat fractionation is efficient to concentrate cryoprotective protein from soybean whey.

A Simple and Rapid Cryopreservation Technique for Ciliates: A Long-Term Storage Procedure Used for Marine Scuticociliates.

Pseudocohnilembus persalinus is a free-living marine scuticociliate that, as a new model organism, has been used in a wide variety of studies. However, long-term laboratory maintenance for this species is mainly achieved by subculture that requires rigorous culture environments and, too often, cultures of the organism die out for a variety of reasons. Successful transport of viable cultures also poses problems for researchers. This study describes a simple and rapid protocol for long-term cryopreservation of P. persalinus. The effects of physiological states of individuals before freezing, the type and concentration of cryoprotectant, and optimal temperatures for freezing and thawing were assessed. A cryopreservation protocol, using a mixture of 30% glycerol and 70% concentrated P. persalinus cell culture, incorporating rate-controlled freezing at -80 °C before liquid nitrogen storage, maintained a high recovery efficiency after 8 wk of storage. These results suggest that broader application of this protocol to build a cryopreserved marine protozoa culture bank for biological studies may be possible.

First birth of a healthy infant following intra-cytoplasmic sperm injection using a new permeable cryoprotectant-free sperm vitrification protocol.

PURPOSE: The purpose of this study is to present the first birth of healthy infant born following ICSI using the new permeable cryoprotectant-free sperm vitrification protocol Easy-Sperm®. PRINCIPAL RESULTS: A 39 years old woman and his 40 years old partner underwent egg donation treatment at IVF-Spain Alicante (Spain). Half of the mature oocytes obtained from a young and healthy donor were fertilized by ICSI, using slow-frozen spermatozoa and the other half with vitrified spermatozoa. A total of 5 blastocysts were obtained on day 5 (3 resulting from vitrified spermatozoa and 2 from frozen sperm). The best embryo, with AA quality (derived from one of the oocytes fertilized with vitrified sperm) was transferred. The woman conceived and, following a normal pregnancy, delivered a healthy boy. CONCLUSIONS: To the best of our knowledge, this is the first case report of a successful pregnancy and delivery of a healthy infant from ICSI with permeable vitrified spermatozoa in an oocyte donation program with transfer on blastocyst stage.

Marker-Independent In Situ Quantitative Assessment of Residual Cryoprotectants in Cardiac Tissues.

Cryomedium toxicity is a major safety concern when transplanting cryopreserved organs. Therefore, thorough removal of potentially toxic cryoprotective agents (CPAs) is required before transplantation. CPAs such as dimethyl-sulfoxide (DMSO), propylene glycol (PG), and formamide (FMD), routinely employed in ice-free cryopreservation (IFC), have advantages in long-term preservation of tissue structures compared with conventional cryopreservation employing lower CPA concentrations. This study evaluated the impact of potential residual CPAs on human cardiac valves. Raman microspectroscopy and Raman imaging were established as nondestructive marker-independent techniques for in situ quantitative assessment of CPA residues in IFC valve tissues. In detail, IFC valve leaflets and supernatants of the washing solutions were analyzed to determine the washing efficiency. A calibration model was developed according to the CPA's characteristic Raman signals to quantify DMSO, PG and FMD concentrations in the supernatants. Single point Raman measurements were performed on the intact tissues to analyze penetration properties. In addition, Raman imaging was utilized to visualize potential CPA residues. Our data showed that washing decreased the CPA concentration in the final washing solution by 99%, and no residues could be detected in the washed tissues, validating the multistep CPA removal protocol routinely used for IFC valves. Raman analysis of unwashed tissues showed different permeation characteristics depending on each CPA and their concentration. Our results demonstrate a great potential of Raman microspectroscopy and Raman imaging as marker-independent in situ tissue quality control tools with the ability to assess the presence and concentration of different chemical agents or drugs in preimplantation tissues.

Isochoric conditions enable high subfreezing temperature pancreatic islet preservation without osmotic cryoprotective agents.

While biological systems are typically studied under isobaric (constant pressure) conditions, recent reports on the bio-thermodynamics of isochoric (constant volume) systems point to their potential for subfreezing-temperature preservation of biological matter. This preliminary study, in which we report that pancreatic islets can survive multi-day preservation at high subfreezing temperatures in an isochoric chamber without osmotic cryoprotective agents (CPA), highlights the potential of isochoric cryopreservation in an application of clinical value.

Simultaneous monitoring of different vitrification solution components permeating into tissues.

Cryopreservation can be used for long-term preservation of tissues and organs. It relies on using complex mixtures of cryoprotective agents (CPAs) to reduce the damaging effects of freezing, but care should be taken to avoid toxic effects of CPAs themselves. In order to rationally design cryopreservation strategies for tissues, it is important to precisely determine permeation kinetics of the protectants that are used to ensure maximum permeation, while minimizing the exposure time and toxicity effects. This is particularly challenging with protectant solutions consisting of multiple components each with different physical properties and diffusing at a different rate. In this study, we show that an attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) setup can be used to simultaneously monitor diffusion of multiple components in a mixture into tissues in real time. Diffusion studies were done with decellularized heart valves using a sucrose-DMSO mixture as well as vitrification solution VS83. To assess diffusion kinetics of different solutes in mixtures, the increase in specific infrared absorbance bands was monitored during diffusion through the tissue. Solute specific wavenumber ranges were selected, and the calculated area was assumed to be proportional to the CPA concentration in the tissue. A diffusion equation based on Fick's second law of diffusion fitted the experimental data quite well, and clear differences in permeation rates were observed among the different mixture components dependent on molecular size and physical properties.

Preservation of Domesticated Honey Bee (Hymenoptera: Apidae) Drone Semen.

Preservation of honey bee (Apis mellifera L., Hymenoptera: Apidae) sperm, coupled with instrumental insemination, is an effective strategy to protect the species and their genetic diversity. Our overall objective is to develop a method of drone semen preservation; therefore, two experiments were conducted. Hypothesis 1 was that cryopreservation (-196 °C) of drone semen is more effective for long-term storage than at 16 °C. Our results show that after 1 yr of storage, frozen sperm viability was higher than at 16 °C, showing that cryopreservation is necessary to conserve semen. However, the cryoprotectant used for drone sperm freezing, dimethyl sulfoxide (DMSO), can harm the queen and reduce fertility after instrumental insemination. Hypothesis 2 was that centrifugation of cryopreserved semen to reduce DMSO prior to insemination optimize sperm quality. Our results indicate that centrifuging cryopreserved sperm to remove cryoprotectant does not affect queen survival, spermathecal sperm count, or sperm viability. Although these data do not indicate that centrifugation of frozen-thawed sperm improves queen health and fertility after instrumental insemination, we demonstrate that cryopreservation is achievable, and it is better for long-term sperm storage than above-freezing temperatures for duration of close to a year.

Determination of residual dimethylsulfoxide in cryopreserved cardiovascular allografts.

Dimethylsulfoxide (DMSO) is a solvent which protects the structure of allografts during the cryopreservation and thawing process. However, several toxic effects of DMSO in patients after transplantation of cryopreserved allografts have been described. The aim of this study is to determine the residual DMSO in the cardiovascular allografts after thawing and preparation of cryopreserved allografts for clinical application following guidelines of the European Pharmacopoeia for DMSO detection. Four types of EHB allografts (aortic valve-AV, pulmonary valve-PV, descending thoracic aorta-DA, and femoral artery-FA) are cryopreserved using as cryoprotecting solution a 10% of DMSO in medium 199. Sampling is carried out after thawing, after DMSO dilution and after delay of 30 min from final dilution (estimated delay until allograft implantation). After progressive thawing in sterile water bath at 37-42 °C (duration of about 20 min), DMSO dilution is carried out by adding consecutively 33, 66 and 200 mL of saline. Finally, tissues are transferred into 200 mL of a new physiologic solution. Allograft samples are analysed for determination of the residual DSMO concentration using a validated Gas Chromatography analysis. Femoral arteries showed the most important DMSO reduction after the estimated delay: 92.97% of decrease in the cryoprotectant final amount while a final reduction of 72.30, 72.04 and 76.29% in DMSO content for AV, PV and DA, was found, respectively. The residual DMSO in the allografts at the moment of implantation represents a final dose of 1.95, 1.06, 1.74 and 0.26 mg kg-1 in AV, PV, DA and FA, respectively, for men, and 2.43, 1.33, 2.17 and 0.33 mg kg-1 for same tissues for women (average weight of 75 kg in men, and 60 kg in women). These results are seriously below the maximum recommended dose of 1 g DMSO kg-1 (Regan et al. in Transfusion 50:2670-2675, 2010) of weight of the patient guaranteeing the safety and quality of allografts.

Supercooling capacity and cryoprotectants of overwintering larvae from different populations of Holcocerus hippophaecolus.

BACKGROUND: Holcocerus hippophaecolus is the most serious pest occurred in seabuckthorn forest of three north areas. OBJECTIVE: The primary aims of the current study were to explore the physiological mechanisms and adaptability of H. hippophaecolus to low temperatures. MATERIALS AND METHODS: Assessing supercooling point, freezing point, and cryoprotectants of different larval instars from three different populations. RESULTS: Supercooling capacity of larvae from the 8-13 instar groups was relatively independent of temperature and other indicators such as latitude. Larvae from the 14-16 instar groups were sensitive to temperature and latitude, with generally lower limits and a wider range of SCPs than those of the other instar groups. CONCLUSION: For each population, the differences in the supercooling capacity of different instar stages for the identical period were not significant. The metabolism of fat and glycogen might not be the primary factors affecting the supercooling capacity.

Evaluación de la motilidad espermática usando dos crioprotectores / Evaluation of sperm motility using two cryoprotectants

La infertilidad, es un problema que afecta a una gran cantidad de parejas. Una de sus causas es la disminución de la calidad seminal debido, por ejemplo, a tratamientos gonadotóxicos. La criopreservación seminal es la técnica que permite conservar y almacenar espermatozoides sin que pierdan su capacidad fecundante; siendo esta una herramienta fundamental en reproducción asistida. El objetivo de este trabajo ha sido optimizar la técnica de criopreservación. Para ello se llevó a cabo un estudio, sobre muestras de pacientes en estudio por problemas de fertilidad, en el que se compararon dos medios de criopreservación (SpermCryo™All-round y CryoSperm™) y la aplicación o no de un baño en nitrógeno líquido a las muestras (previo a su almacenamiento); así como el efecto del tiempo que transcurre desde la eyaculación hasta el procesado sobre la calidad de la muestra. Las posibles variaciones fueron estudiadas con un analizador automático, mediante la realización de test pre- y post-congelación para comprobarla movilidad espermática

Infertility is a problem that affects a lot of couples. One of its causes is a decreased semen quality due to, for example, gonadotoxic treatments. The cryopreservation of human semen is the technique that allows sperm preserving and storing without losing their fertilizing capacity; being a fundamental tool in assisted reproduction. The aim of this study was to optimize the cryopreservation technique. To this end, a study carried out on samples of patients under study by fertility problems, in which two cryoprotectant media (SpermCryo™ All-round and CryoSperm™) and the execution or non-execution of an immersion of the samples in liquid nitrogen (before storage) were compared; and the effect of the time between ejaculation and the processing on the quality of the sample. Variations were studied with an automatic analyzer by performing pre- and post-thaw sperm motility tests. The results show no difference between the two cryoprotectants media, but seems to have a tendency to obtain better postthaw mobility with either depending on sample characteristics. Moreover, the liquid nitrogen bath had no apparent effects on post-thaw results. However, we must highlight the importance of time in the processing of semen samples once liquefied, to avoid decreased sperm quality. To improve post-thaw outcomes the key lies in the necessity to adjust the freezing protocol to the sample characteristics and a correct implementation of the protocol cryopreservation (selection and addition of cryoprotectant media...); favoring the management of infertility and the success of assisted reproduction techniques

Freeze-drying of ampicillin solid lipid nanoparticles using mannitol as cryoprotectant

abstract Solid lipid nanoparticles (SLNs) are interesting colloidal drug-delivery systems, since they have all the advantages of the lipid and polymeric nanoparticles. Freeze-drying is a widely used process for improving the stability of SLNs. Cryoprotectants have been used to decrease SLN aggregations during freeze-drying. In this study Ampicillin was chosen to be loaded in a cholesterol carrier with nano size range. To support the stability of SLNs, freeze-drying was done using mannitol. Particle size, drug release profile and antibacterial effects were studied after freeze-drying in comparison with primary SLNs. Preparations with 5% mannitol showed the least particle size enlargement. The average particle size was 150 and 187 nm before and after freeze-drying, respectively. Freeze-drying did not affect the release profile of drug loaded nanopartilces. Also our study showed that lyophilization did not change the antimicrobial effect of ampicillin SLNs. DSC analysis showed probability of chemical interaction between ampicillin and cholesterol.

resumo Nanoparticulas lipídicas sólidas (NLSs) são sistemas coloidais de liberação interessantes, uma vez que reúnem todas as vantagens de nanopartículas lipídicas e poliméricas. A liofilização é um processo amplamente utilizado para melhorar a estabilidade das NLSs e os crioprotetores têm sido usados para diminuir a agregação destas durante esse processo. Neste estudo, a ampicilina foi escolhida para ser encapsulada em um carreador de colesterol de escala nanométrica. Para manter a estabilidade das NLSs, a liofilização foi realizada utilizando-se manitol. O tamanho de partícula, o perfil de liberação do fármaco e os efeitos antibacterianos foram estudados após a liofilização em comparação com a NLSs primária. De acordo com os resultados, as preparações que contêm 5% de manitol mostraram o menor aumento do tamanho de partícula. Os resultados de tamanhos médio foram de 150 e 187 nm antes e depois da liofilização, respectivamente. O perfil de liberação prolongada, bem como o efeito antimicrobiano da ampicilina NLSs não foram alterados após a liofilização. A análise por DSC evidenciou provável interação entre a ampicilina e o colesterol.

Aloe vera na criopreservação do sêmen de tambaqui (Colossoma macropomum) / Aloe vera in the cryopreservation of tambaqui (Colossoma macropomum) sperm

This study aimed to evaluate the extract of Aloe vera (AV) associated or not with 10% Dimethylsulfoxide (DMSO) in cryopreservation of tambaqui semen. For the formation of the pools (n= 14), 30 males were hormonally induced twice. Each pool had the objective motility, curvilinear velocity, straight-line velocity, average path velocity and morphology analyzed before and after cryopreservation of semen. The means for cryopreservation were constituted of Powder Coconut Water-104 diluent added DMSO and/or AV (5 or 10%). After cryopreservation, motility, velocities and morphology were reduced significantly when compared to fresh semen. For sperm motility the best treatment was that using only DMSO (20,86±8,31) and DMSO + 5% AV (15.71±9.77). For the velocities, the worse treatment was DMSO+10% AV. Treatment with only the addition of DMSO had a significantly higher effect than others on percentage of morphologically normal sperm. The mean correlation found was between motilityand the rate of morphologically normal sperm (r = 0.687). In conclusion, the addition of AV does not provide greater protection for spermatozoa during cryopreservation.

Evaluation of cryoprotective effect of Turkish pine honey on common carp (Cyprinus carpio) spermatozoa.

BACKGROUND: The cryopreservation procedures that allow preserving sperm cells have been applied for sperm of many species. A sugar like glucose, fructose and sucrose were frequently used in cryomedia but up to the present pine honey was not used for cryopreservation of sperm cells. OBJECTIVE: The objective of present study is to investigate the effect of pine honey in various concentrations of 100, 200, 300, 400 and 500 mg/ml solutions on cryopreservation and fertilization ability of spermatozoa of common carp (Cyprinus carpio). MATERIALS AND METHODS: Totally 12.5 % (v/v) Me2SO as a cryoprotectant and 10 % (v/v) egg yolk added all extenders. Pine honey also compared with sugars as glucose, fructose (monosaccharide) and sucrose (disaccharide). Collected semen samples were diluted at the ratio of 1:9 with the extenders. After dilutions, the sperm motility was assessed for each group and then the diluted semen samples were cryopreserved. RESULTS: The extenders containing 300 mg ml-1 pine honey group showed both highest post thaw motility 75.3 ± 5.1 %, motility duration (s) 47.3 ± 2.5 % and hatching ratio 42.6 ± 4.2 % than other cryopreserved groups (P<0.05). CONCLUSION: Using the pine honey in cryomedia is effective for cryopreservation especially about hatching success of egg fertilized by frozen-thawed sperm of common carp.

Evaluation of intracellular and extracellular trehalose as a cryoprotectant of stem cells obtained from umbilical cord blood.

Cord blood is a source of hematopoietic stem cells used in transplantation in which hematopoietic reconstitution is necessary. This transplant modality requires the cryopreservation of hematopoietic stem cells (HSCs). Dimethyl sulfoxide has been used as a cryoprotectant (CPA) in the cryopreservation of HSCs; however, it has been demonstrated that Me2SO exhibits toxic side effects to the human body. Due to its stability upon freezing, disaccharides such as trehalose have been investigated as a cryoprotectant. This study investigated the hypothesis that a cryopreservation solution containing intracellular and extracellular trehalose improves the recovery of stem cells after cryopreservation. After thawing, the cells were tested for their viability using the 7AAD stain, CD45+/CD34+ cells were assessed using flow cytometry and the MTT viability assay, and the proportion of hematopoietic progenitor cells was measured using the CFU assay. Our results showed the effectiveness of the solution containing intracellular and extracellular trehalose in the cryopreservation of cord blood cells, demonstrating that trehalose may be an optimal cryoprotectant when present both inside and outside of cells.

Effect of addition of coconut water (Cocos nucifera) to the freezing media on post-thaw viability of boar sperm.

The aims of this experiment were to evaluate the addition of coconut water in natura to the freezing media, compare the effect of deionized water vs filtered water of coconut over the post-thaw seminal characteristics, and evaluate the effect of the deionized water and in natura coconut water on the seminal characteristics of boar sperm at different post-thaw times. Thirty-four ejaculates were used divided in three aliquots which received one of the following treatments (T): T1, LEY (bidistilled water, lactose, and egg yolk) and LEYGO (LEY + glycerol and Orvus ET paste); T2, LEY(A) (coconut deionized water, lactose, and egg yolk)-LEYGO(A); and T3, LEY(B) (in natura coconut water, lactose, and egg yolk)-LEYGO(B). Samples of boar semen were frozen according to the Westendorf method, thawed at 38°C, and evaluated at three incubation times (0, 30, and 60 min). Seminal characteristics assessed were motility (Mot), acrosomal integrity (AInt), membrane integrity (MInt), and mitochondrial activity (MAct). T1 showed a higher percentage of viable sperm than T3 (Mot 36.5 vs 5.4 %, AInt 61.8 vs 41.2 %, MInt 50.4 vs 41.3 %, and MAct 56.9 vs 50.5 %). T2 kept a higher percentage of viable sperm at all incubation times. In natura coconut water showed a detrimental effect over the viability of the frozen-thawed boar semen. Deionized coconut water improved the boar semen viability post-thaw, outperforming results of in natura coconut water.