RESUMO
Semen cryopreservation is a very important technique for assisted reproduction; however, the cryopreservation process is harmful because it results in a reduction in sperm motility and viability, and leads to premature signals of capacitation, resulting in lesser than desirable fertility rates after artificial insemination. A fraction of seminal plasma, enriched in proteins that contain type II fibronectin domains (FNII) can reverse molecular indicators of cryo-capacitation. The beneficial effects of these proteins, however, depend on the relative abundance in seminal plasma. To create a safe additive for improving frozen sperm functionality, in the present study there was cloning and expression of a recombinant peptide containing four FNII domains (named TrxA-FNIIx4-His6) and evaluation of its effect after addition to frozen/thawed ram sperm. The cDNA for this protein was expressed in E. coli and after denaturation and re-naturalization of the protein, toxicity and binding capacity were assessed. By fluorescent labelling assessment, there was binding of the protein to the thawed sperm. At the two doses used (0.15 and 0.3 µM), TrxA-FNIIx4-His6 had the capacity to reverse the molecular indicators of cryo-capacitation as indicated by the reduction on phosphorylated substrates of PKA. Furthermore, the supplementation with this protein resulted in a normal capacitation process as evidenced by the increase in the in vitro fertilization rate when the greatest concentration of the protein was evaluated (73.25⯱â¯2.95; 40.13⯱â¯11.82 for 0.3 µM and control, respectively). There was no effect of protein supplementation on sperm objective motility compared to untreated sperm. In conclusion, the use of TrxA-FNIIx4-His6 is a promising biotechnological approach for cryopreserving ram sperm and maintaining sperm viability.
Assuntos
Criopreservação/veterinária , Fertilização in vitro/veterinária , Peptídeos/farmacologia , Proteínas de Plasma Seminal/farmacologia , Capacitação Espermática/efeitos dos fármacos , Animais , Clonagem Molecular , Criopreservação/métodos , Crioprotetores/química , Crioprotetores/metabolismo , Crioprotetores/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Fertilização in vitro/métodos , Fibronectinas/química , Fibronectinas/genética , Inseminação Artificial/métodos , Inseminação Artificial/veterinária , Masculino , Peptídeos/genética , Domínios Proteicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Análise do Sêmen/veterinária , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Proteínas de Plasma Seminal/química , Proteínas de Plasma Seminal/genética , Ovinos , Capacitação Espermática/fisiologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacosRESUMO
Boar semen cannot be immediately cryopreserved, it need be hold at 17⯰C prior to cryopreservation, holding time has been used to improve cryopreserved boar semen, since holding time allows a prolonged interaction between spermatozoa and seminal plasma components. However, until now only few periods of holding time have been studied, and boar semen had been held at 17⯰C for 24â¯h to facilitate its manufacture. Thus, this experiment aims to study the effect several holding time (0, 4, 8, 12, 24, 28 and 32â¯h) on boar spermatozoa post-thawed (PT) characteristics. Fifteen sperm-rich fractions of ejaculate were extended in Beltsville Thawing Solution and storage at 17⯰C. After each holding time (0, 4, 8, 12, 24, 28 and 32â¯h), a sample was centrifuged, and sperm pellet was diluted in an extender composed of sugars, amino acids, buffers, 20% egg yolk (v/v), antibiotics, 2% glycerol as a cryoprotectant, and 2% methylformamide (v/v). Cryopreservation was performed with an automatic cryopreservation system. Cryopreserved boar semen was evaluated to spermatozoa kinetics, plasma and acrosomal membranes integrity, mitochondrial membrane potential, detection of superoxide anion, plasma membrane fluidity, and peroxidation. Twenty-four hours of holding increase total and progressive motility, rapid spermatozoa, and integrity of plasma and acrosome membranes. To mitochondrial membrane potential, 32â¯h is needed. However, holding time was not able to control the superoxide anion amount neither membrane lipid peroxidation, and had no effects on membrane fluidity. Thus, to reach the best results of PT boar semen the ideal holding time is 24â¯h.
Assuntos
Criopreservação/métodos , Análise do Sêmen , Preservação do Sêmen/métodos , Sêmen/fisiologia , Motilidade dos Espermatozoides/fisiologia , Acrossomo/metabolismo , Animais , Membrana Celular/metabolismo , Crioprotetores/metabolismo , Crioprotetores/farmacologia , Masculino , Fluidez de Membrana , Potencial da Membrana Mitocondrial , Suínos , Fatores de TempoRESUMO
Abstract Agaricus subrufescens is a basidiomycete which is studied because of its medicinal and gastronomic importance; however, less attention has been paid to its preservation. This study aimed to evaluate the effect of sucrose addition to substrate and cryotube on the viability of Agaricus subrufescens cryopreserved at -20 °C and at -75 °C for one and two years. Zero, 10% or 20% sucrose was added to potato dextrose agar or wheat grain. The mycelia were cryopreserved in the absence of cryoprotectant or with sucrose solutions at 15%, 30% or 45%. After one or two years at -75 °C or at -20 °C, mycelia were thawed and evaluated about viability, initial time of growth, colony diameter and genomic stability. Cryopreservation at -20 °C is not effective to keep mycelial viability of this fungus. Cryopreservation at -75 °C is effective when sucrose is used in substrates and/or cryotubes. Without sucrose, cryopreservation at -75 °C is effective only when wheat grains are used. Physiological characteristic as mycelial colony diameter is negatively affected when potato dextrose agar is used and unaffected when wheat grain is used after two-year cryopreservation at -75 °C. The fungus genome does not show alteration after two-year cryopreservation at -75 °C.
Assuntos
Agaricus/crescimento & desenvolvimento , Criopreservação/métodos , Crioprotetores/metabolismo , Congelamento , Sementes/microbiologia , Sacarose/metabolismo , Triticum/microbiologia , Agaricus/efeitos da radiação , Instabilidade Genômica/efeitos da radiação , Viabilidade Microbiana/efeitos da radiação , Micélio/crescimento & desenvolvimento , Micélio/efeitos da radiação , Fatores de TempoRESUMO
Abstract Basidiomycetes have several biotechnological and industrial applications such as enzyme production, bioremediation, pharmaceutical and functional food production. Due to climatic features, the preservation of several basidiomycetes is threatened, and to guarantee the preservation of this genetic resource, the development of long-term preservation techniques is necessary once there is no universal protocol for the cryopreservation of basidiomycetes. Cryopreservation is a technique in which microorganisms are submitted to ultralow temperatures. Therefore, this study aimed to collect information on the main conditions for long-term cryopreservation of basidiomycetes in the last 20 years. Scientific articles on cryopreservation of basidiomycetes published from 1997 to 2016, were researched, and only the studies on two intervals of cryopreservation were considered: from 1 to 2 years and for longer than 2 years. The analyzed conditions of basidiomycete cryopreservation were: most studied genera, cryopreservation temperature, substrate, cryoprotectant (and preservation substrate), cryopreservation period, thawing temperature and cultivation medium after thawing, physiological and genetic stability of basidiomycetes after thawing in cryopreservation. In this review, the viability of the main cryopreservation conditions of basidiomycetes studied in the last 20 years are presented and discussed.
Assuntos
Basidiomycota/fisiologia , Criopreservação/métodos , Viabilidade Microbiana/efeitos da radiação , Basidiomycota/efeitos da radiação , Crioprotetores/metabolismo , Meios de Cultura/química , Fatores de TempoRESUMO
Basidiomycetes have several biotechnological and industrial applications such as enzyme production, bioremediation, pharmaceutical and functional food production. Due to climatic features, the preservation of several basidiomycetes is threatened, and to guarantee the preservation of this genetic resource, the development of long-term preservation techniques is necessary once there is no universal protocol for the cryopreservation of basidiomycetes. Cryopreservation is a technique in which microorganisms are submitted to ultralow temperatures. Therefore, this study aimed to collect information on the main conditions for long-term cryopreservation of basidiomycetes in the last 20 years. Scientific articles on cryopreservation of basidiomycetes published from 1997 to 2016, were researched, and only the studies on two intervals of cryopreservation were considered: from 1 to 2 years and for longer than 2 years. The analyzed conditions of basidiomycete cryopreservation were: most studied genera, cryopreservation temperature, substrate, cryoprotectant (and preservation substrate), cryopreservation period, thawing temperature and cultivation medium after thawing, physiological and genetic stability of basidiomycetes after thawing in cryopreservation. In this review, the viability of the main cryopreservation conditions of basidiomycetes studied in the last 20 years are presented and discussed.
Assuntos
Basidiomycota/fisiologia , Criopreservação/métodos , Viabilidade Microbiana/efeitos da radiação , Basidiomycota/efeitos da radiação , Crioprotetores/metabolismo , Meios de Cultura/química , Fatores de TempoRESUMO
Agaricus subrufescens is a basidiomycete which is studied because of its medicinal and gastronomic importance; however, less attention has been paid to its preservation. This study aimed to evaluate the effect of sucrose addition to substrate and cryotube on the viability of Agaricus subrufescens cryopreserved at -20°C and at -75°C for one and two years. Zero, 10% or 20% sucrose was added to potato dextrose agar or wheat grain. The mycelia were cryopreserved in the absence of cryoprotectant or with sucrose solutions at 15%, 30% or 45%. After one or two years at -75°C or at -20°C, mycelia were thawed and evaluated about viability, initial time of growth, colony diameter and genomic stability. Cryopreservation at -20°C is not effective to keep mycelial viability of this fungus. Cryopreservation at -75°C is effective when sucrose is used in substrates and/or cryotubes. Without sucrose, cryopreservation at -75°C is effective only when wheat grains are used. Physiological characteristic as mycelial colony diameter is negatively affected when potato dextrose agar is used and unaffected when wheat grain is used after two-year cryopreservation at -75°C. The fungus genome does not show alteration after two-year cryopreservation at -75°C.
Assuntos
Agaricus/crescimento & desenvolvimento , Criopreservação/métodos , Crioprotetores/metabolismo , Congelamento , Sementes/microbiologia , Sacarose/metabolismo , Triticum/microbiologia , Agaricus/efeitos da radiação , Instabilidade Genômica/efeitos da radiação , Viabilidade Microbiana/efeitos da radiação , Micélio/crescimento & desenvolvimento , Micélio/efeitos da radiação , Fatores de TempoRESUMO
BACKGROUND: The Ehrlich Ascitic Carcinoma (EAC) is an experimental transplantable neoplasm that develops in several species of mice. The maintenance of the tumor occurs in vivo. Thus, freezing the cells would reduce the number of passages between animals, ensuring genetic stability and storage for longs period of experimentation. OBJECTIVE: Search by EAC cryoprotectants. MATERIALS AND METHODS: The combinations of nutrient medium (Tris, hen egg yolk, and DEMEN) and cryoprotective agent (Glicerol, Trehalose and DMSO) on freezing EAC cells and the transplantability after defrosting were evaluated. The cooling was conducted at 2 C/min. until -180 degree C and the thawing by immersion in water at 37 degree C. The transplantability was evaluated from cell inoculation in mice for 14 days. RESULTS: The best results were the associations IA (Cryoprotective agent Glycerol 6 % and medium containing 3.0 % Tris w / v, 1.8 % Citric acid w / v, 1.3 % D-fructose w / v and 20 % hen egg yolk v / v) and IIB (Cryoprotective agent Trehalose 100mM and medium containing 50 % coconut water v / v, 25 % sodium citrate 5 % v / v and 20 % hen egg yolk v / v) with 85.2 % and 55.1 % viable cells, respectively. CONCLUSION: These transplantable cells were efficient for tumor development, therefore demonstrating that this method of cryopreservation is simple and affordable.
Assuntos
Carcinoma de Ehrlich/metabolismo , Criopreservação/métodos , Crioprotetores/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Dimetil Sulfóxido/metabolismo , Gema de Ovo/metabolismo , Glicerol/metabolismo , Masculino , Camundongos , Transplante de Neoplasias , Trealose/metabolismoRESUMO
This study evaluated the effect of the use of hypometabolic TRIS extenders in the presence or the absence of AMPK activators as well as the utilization of high cooling rates in the refrigeration step on the freezability of stallion sperm. Twelve ejaculates were cryopreserved using Botucrio® as a control extender and a basic TRIS extender (HM-0) separately supplemented with 10 mM metformin, 2mM 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR), 2 mM Adenosine monophosphate (AMP), 40 µM compound C AMPK inhibitor or 2 mM AMP+40 µM compound C. Our results showed that the utilization of a hypometabolic TRIS extender supplemented or not with AMP or metformin significantly improves stallion sperm freezability when compared with a commercial extender. Additionally, high cooling rates do not affect stallion sperm quality after cooling and post-thawing. Finally, stallion spermatozoa present several putative AMPK sperm isoforms that do not seem to respond to classical activators, but do respond to the Compound C inhibitor.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Criopreservação/veterinária , Crioprotetores/metabolismo , Cavalos/fisiologia , Preservação do Sêmen/veterinária , Espermatozoides/citologia , Trometamina/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Monofosfato de Adenosina/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Criopreservação/métodos , Hipoglicemiantes/metabolismo , Masculino , Metformina/metabolismo , Ribonucleotídeos/metabolismo , Preservação do Sêmen/métodos , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismoRESUMO
BACKGROUND: Embryo cryopreservation has been used for the creation of genetic banks with diploid resources, and among different techniques, vitrification is considered as the most promising method. OBJECTIVE: The goal is to evaluate the major aspects of the existing vitrification techniques and to evaluate their efficacy in terms of embryo morphology. METHODS: Electronic searches in the PubMed and ScienceDirect databases were performed with the keyword combination: fish, embryo and vitrification. Pubmed retrieved 26 articles and Science Direct resulted in 464 articles. For this review, only studies that developed and tested vitrification protocols in fish embryos were included. Research regarding cryoprotectant toxicity and permeability were excluded. There were no restrictions on publication date or language. With these criteria, a total of ten articles were evaluated. RESULTS: In these articles, the major aspects to be considered for the development of new vitrification protocols are: the cryoprotectants' toxicity, the embryos' development stage, the exposure to and the permeability of the cryoprotectants, vitrification devices and vitrification-warning cycle. CONCLUSION: The survival were limited, however, the preservation of embryonic morphology after thawing indicates the possibility of preserving fish embryos via the vitrification technique.
Assuntos
Criopreservação/métodos , Criopreservação/veterinária , Crioprotetores/metabolismo , Embrião não Mamífero/fisiologia , Peixes/embriologia , Vitrificação , Animais , Criopreservação/instrumentação , Crioprotetores/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/embriologiaRESUMO
BACKGROUND: CIP maintains the largest in vitro clonal potato collection in the world, comprising 4,013 landraces and 3,353 improved accessions. The in vitro technology is more efficient and secure than conservation in the field, allowing in vitro plantlets to be stored for approximately 2 years without sub-culture. This method however is not ideal for the long-term germplasm conservation because it is labor consuming, costly, and carries risks of losing accessions due to human error, such as contamination and mislabeling during sub-culturing. OBJECTIVE: To improve the potato cryopreservation procedure based on the droplet PVS2 vitrification. METHODS: The improved method is as follows: excision of 1.8-2.5 mm apical shoot tips from 3 weeks old cultures; 15 min exposure to a loading solution and 50 min to PVS2 (at 0 degree C); ultra-rapid cooling on aluminum foil strips (0.5 x 2 cm) in LN; rewarming (20 min) in 1.2 M sucrose MS liquid medium; post-cryo culture in the dark on potato meristem medium with progressively decreased sucrose levels (daily transfers from 0.3, to 0.2, to 0.1 M and maintained on 0.07 M). This method was compared with those previously applied by IPK (Germany) and CIP potato genebanks. RESULTS: Survival and recovery were higher using the PVS2 droplet method. Cultivars from several species, one frost tolerant (Solanum juzecpzukii, cv. Pinaza) and two drought tolerant (S. tuberosum subsp andigena, cv Ccompis, and Solanum spp, cv Desiree) responded similarly. CONCLUSIONS: The improved method is recommended for the long term conservation of diverse potato germplasm.
Assuntos
Criopreservação/métodos , Brotos de Planta/fisiologia , Solanum tuberosum/fisiologia , Vitrificação , Crioprotetores/metabolismo , Dimetil Sulfóxido/metabolismo , Sacarose/metabolismoRESUMO
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.
Assuntos
Criopreservação , Crioprotetores/metabolismo , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Trealose/metabolismo , Sobrevivência Celular , Células Cultivadas , Criopreservação/métodos , Crioprotetores/administração & dosagem , Crioprotetores/análise , Feminino , Células-Tronco Hematopoéticas/metabolismo , Humanos , Lipossomos , Trealose/administração & dosagem , Trealose/análiseRESUMO
The aim of this work was to evaluate the protective effect of catalase (CAT) on frozen/thawed ibex epididymal sperm recovered post mortem, and to detect any harmful effect this might have on sperm fertilisation capacity. Epididymal spermatozoa were diluted using a Tris-citric acid-glucose medium (TCG) composed of 3.8% Tris (w/v), 2.2% citric acid (w/v), 0.6% glucose (w/v), 5% glycerol (v/v), and 6% egg yolk (v/v). Sperm masses from the right epididymis were diluted with TCG medium, while those from the left were diluted with TCG medium supplemented with 200IU/mL CAT. Heterologous in vitro fertilisation (IVF) was used to assess the fertilisation capacity of this sperm. The addition of CAT to the extender did not improve frozen/thawed sperm variables. Moreover, a reduced fertilisation capacity was detected: sperm diluted with TCG provided 25.5% 2PN zygotes, while just 13.2% was recorded for that diluted with TCG-CAT (P<0.01). The percentage of cleaved embryos at 48hpi was higher (P<0.01) with the TCG sperm than with the TCG-CAT sperm (16.7% vs. 7.6%). The use of 200IU/mL CAT as an additive cannot, therefore, be recommended for the preservation of ibex epididymal sperm. Other antioxidants should, however, be tested in both this and related wild mountain ungulates.
Assuntos
Catalase/metabolismo , Criopreservação/veterinária , Fertilização in vitro/veterinária , Cabras/fisiologia , Preservação do Sêmen/veterinária , Espermatozoides/citologia , Animais , Antioxidantes/metabolismo , Criopreservação/métodos , Crioprotetores/metabolismo , Epididimo/citologia , Feminino , Fertilização in vitro/métodos , Masculino , Preservação do Sêmen/métodos , Espermatozoides/metabolismoRESUMO
Cryopreservation of plant species is poorly investigated in Brazil. The aim of this study was to cryopreserve Byrsonima intermedia shoot apical meristems through droplet vitrification. A culture medium for shoot-tips growth was established using the Woody Plant Medium supplemented with 2.22 uM 6-benzylaminopurine. Excised shoot-tips were subjected to pre-culture and/or post-culture treatments on Murashige and Skoog medium with 0.3 M sucrose for 24 h prior dehydration on PVS2 at 0°C for 15, 30 or 45 minutes prior to plunging in liquid nitrogen. The effect of 15 days of shoot pre-growth on a high osmotic medium (0.3 M sucrose or 0.21 M sorbitol + 0.09 M sucrose) prior to meristem excision and cryopreservation was also investigated. Pre-culturing shoot-tips on 0.3 M sucrose for 24 h prior to cryopreservation increased the regrowth level after thawing to 90%. Shoot-tips excised from shoots pre-grown on MS + 0.21 M sorbitol + 0.09 M sucrose for 15 days presented a satisfactory regrowth level (67%).
Assuntos
Criopreservação/métodos , Malpighiaceae/crescimento & desenvolvimento , Brotos de Planta/crescimento & desenvolvimento , Vitrificação , Compostos de Benzil , Crioprotetores/metabolismo , Cinetina/metabolismo , Malpighiaceae/efeitos dos fármacos , Meristema/efeitos dos fármacos , Meristema/crescimento & desenvolvimento , Brotos de Planta/efeitos dos fármacos , Plantas Medicinais/efeitos dos fármacos , Plantas Medicinais/crescimento & desenvolvimento , Purinas , Sorbitol/metabolismo , Sacarose/metabolismoRESUMO
CSF470 vaccine is a mixture of four lethally irradiated melanoma cell lines, administered with BCG and GM-CSF, which is currently being tested in a Phase II/III Clinical trial in stage II/III melanoma patients. To prepare vaccine doses, irradiated melanoma cell lines are frozen using dimethyl sulfoxide (Me(2)SO) and stored in liquid nitrogen (liqN(2)). Prior to inoculation, doses must be thawed, washed to remove Me(2)SO and suspended for clinical administration. Avoiding the use of Me(2)SO and storage in liqN(2) would allow future freeze-drying of CSF470 vaccine to facilitate pharmaceutical production and distribution. We worked on the development of an alternative cryopreservation methodology while keeping the vaccine's biological and immunogenic properties. We tested different freezing media containing trehalose suitable to remain as excipients in a freeze-dried product, to cryopreserve melanoma cells either before or after gamma irradiation. Melanoma cells incorporated trehalose after 5 h incubation at 37°C by fluid-phase endocytosis, reaching an intracellular concentration that varied between 70-140 mM depending on the cell line. Optimal freezing conditions were 0.2 M trehalose and 30 mg/ml human serum albumin, at -84°C. Vaccine doses could be frozen in trehalose at -84°C for at least four months keeping their cellular integrity, antigen expression and apoptosis/necrosis profile after gamma-irradiation as compared to Me(2)SO control. Non-irradiated melanoma cell lines also showed comparable proliferative capacity after both cryopreservation procedures. Trehalose-freezing medium allowed us to cryopreserve melanoma cells, either alive or after gamma irradiation, at -84°C avoiding the use of Me(2)SO and liqN(2) storage. These cryopreservation conditions could be suitable for future freeze-drying of CSF470 vaccine.
Assuntos
Vacinas Anticâncer/metabolismo , Linhagem Celular Tumoral/citologia , Criopreservação/métodos , Melanoma/prevenção & controle , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/efeitos da radiação , Crioprotetores/metabolismo , Dimetil Sulfóxido/metabolismo , Congelamento , Humanos , Melanoma/metabolismo , Trealose/metabolismoRESUMO
Even though sheep embryo cryopreservation is a commonly used procedure the survival and pregnancy outcomes can vary greatly. This study investigated whether cryopreservation was causing subtle changes in ultrastructure, mitochondrial activity or cytoskeletal integrity. Sheep embryos were either slow cooled in 1.5 M EG (n = 22), or vitrified in 20% EG + 20% DMSO with 0.5 M sucrose in Open Pulled Straws (OPS) (n = 24). One hour after warming the cryopreserved embryos differed from control embryos in that they had no mitochondrial activity combined with cytoskeletal disorganization and large vesicles. Vitrified embryos also showed many points of cytoskeleton disruption. Ultrastructural alterations resulting from actin filaments disorganization were observed in both cryopreserved groups. This includes areas presenting no cytoplasmic organelles, Golgi complex located far from the nucleus and a decrease of specialized intercellular junctions. Additionally, large vesicles were observed in vitrified morulae and early blastocysts. The alterations after cryopreservation were proportional to embryo quality as assessed using the stereomicroscope. Even in the absence of mitochondrial activity, grade I and II cryopreserved embryos contained mitochondria with normal ultrastructure. Embryos classified as grade I or II in the stereomicroscope revealed mild ultrastructural alterations, meaning that this tool is efficient to evaluate embryos after cryopreservation.
Assuntos
Criopreservação/veterinária , Embrião de Mamíferos/ultraestrutura , Ovinos/embriologia , Vitrificação , Animais , Criopreservação/métodos , Crioprotetores/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Dimetil Sulfóxido/metabolismo , Embrião de Mamíferos/metabolismo , Etilenoglicol/metabolismo , Mitocôndrias/metabolismoRESUMO
The aims of this study were to determine the stability of Podoviridae coliphage CA933P during lyophilization and storage in different media, and to establish similarities between the results obtained and those expected through mechanisms described for proteins stabilization during freeze-drying. PBS and SM buffer were assayed as lyophilization media. The effect of inorganic salts concentration as well as the addition of disaccharides on phage stability during freeze-drying and storage was also studied. The addition of low sucrose concentration (0.1 mol l⻹) to SM buffer stabilized phage during freezing and drying steps of the lyophilization process, but higher sugar concentrations were detrimental to phage stability during freeze-drying. Sucrose stabilized phage during storage for at least 120 days. The lyoprotective effect of low concentrations of disaccharides during the drying step of the lyophilization of proteins as well as the stabilization of the freeze-dried product in time correlated with the results obtained for phage CA933P.
Assuntos
Crioprotetores/metabolismo , Liofilização/métodos , Podoviridae/fisiologia , Sacarose/metabolismo , Soluções TampãoRESUMO
The tambaqui is an Amazonian fish of great economic and environmental importance to Brazil and other South American countries. Several semen cryopreservation methodologies have been tested for different Brazilian fish species; however, there is little information on the use of this technique on tambaqui semen. The aim of the present study was to investigate the effect of osmolarity and activation solutions on sperm kinetics and, glucose solutions, cryoprotectants, dilution ratios, egg yolk and freezing methods on tambaqui semen freezing. The osmolarity of 230 mOsm was suitable for simultaneously yielding higher sperm motility (85%) and motility time (54 sec.) and osmolarities above 360 mOsm maintain immobile tambaqui sperm. The tambaqui semen can be successfully cryopreserved when diluted 1:9 in freezing medium composed of 5 percent glucose solution (290 mOsm) with 10 percent methylglycol and 5 percent egg yolk, and frozen directly in a dry shipper container.
Assuntos
Caraciformes/fisiologia , Preservação do Sêmen/veterinária , Espermatozoides/citologia , Animais , Crioprotetores/metabolismo , Gema de Ovo/metabolismo , Congelamento , Glucose/metabolismo , Masculino , Concentração Osmolar , Preservação do Sêmen/métodos , Motilidade dos Espermatozoides , Espermatozoides/metabolismoRESUMO
Extenders with a defined composition containing only components with clearly protective effects on sperm during storage would be an advantage. The aims of the present work were to assess whether caseinate, improves cooled and frozen equine semen quality. Semen from six stallions were suspended with four different cooling extenders C1) Kenney extender; C2) 0.6 % caseinate; C3) 2.7 % caseinate ; and C4) C1 + 2.1 % caseinate, and frozen extenders: F1) INRA 82 extender; F2) 1.35 % caseinate; and F3) 2.7 % caseinate. Although there was no significant difference between the motility rate among the cooled (C1:45.0, C2:36.7, C3:38.3 and C4:48.3) and frozen extenders (F1:16.9, F2:21.1 and F3:18.6), significant higher values of sperm velocity variables were observed with the 1.35 % caseinate extender compared to the control (VSL: 40.8 x 18.9 and VAP: 46.8 x 25.0 µm/s), respectively. Caseinate seemed to be responsible for sperm protection during preservation and showed to be as efficient as milk.
Assuntos
Caseínas , Criopreservação/veterinária , Crioprotetores , Preservação do Sêmen/veterinária , Espermatozoides/citologia , Animais , Caseínas/metabolismo , Temperatura Baixa , Criopreservação/métodos , Crioprotetores/metabolismo , Cavalos , Masculino , Preservação do Sêmen/métodos , Motilidade dos Espermatozoides , Espermatozoides/metabolismoRESUMO
Cryopreservation of fish gametes has been studied extensively in the last few decades, but the successful cryopreservation of fish embryos remains elusive. However, recent studies using short-term chilling techniques have shown that it is possible to store embryos at low temperatures with no significant loss in viability. Information on cryopreservation of Neotropical freshwater fish embryos has so far been very limited in the literature. In the present study, chilling protocols for storage of pacu embryos at -8°C for up to 24 h were studied using different concentrations of sucrose in methanol. Embryos tolerated the subzero temperature for up to 6 h with no adverse effects (P > 0.05). After 12 h chilling, hatching rate of 64.0 +/- 3.5 percent was recorded. Low temperature storage of pacu embryos by chilling is detailed here for the first time. Further studies are needed to extend the storage time and to improve the hatching rate.
Assuntos
Criopreservação/métodos , Crioprotetores/metabolismo , Embrião não Mamífero/fisiologia , Peixes/embriologia , Sacarose/metabolismo , Animais , Embrião não Mamífero/embriologia , Embrião não Mamífero/ultraestrutura , Pesqueiros , Metanol/metabolismoRESUMO
The current use of ingredients of animal origin, such as egg yolk, in semen extenders presents a risk of microbial contamination, and has led to the search for alternatives. Such an extender is commercially available for bull semen (Bioexcell), IMV, L'Aigle, France), and it has previously been tested in vitro for freezing ram semen, with satisfactory results. The aim of the present study was to compare the fertility results of ewes in Uruguay, after cervical insemination with ram semen that was frozen in Bioexcell versus semen frozen in a conventional milk-egg yolk extender (control). Semen from five Corriedale rams was frozen, using a split sample design, in either milk-egg yolk or Bioexcell extender, using a two-step extension method. The sperm parameters assessed after thawing were subjective motility, membrane integrity (SYBR-14/PI), and capacitation status (CTC). Thawed semen was inseminated intracervically once during spontaneous estrus in 970 Corriedale ewes that grazed in natural pastures, under extensive management conditions. Fertility was recorded as nonreturn rates at 21 days (NRR-21) and 36 days (NRR-36) after artificial insemination (AI), as well as pregnancy rate (PR-US, diagnosed ultrasonographically 50 days after AI of the last ewe). Subjective motility was slightly higher in Bioexcell than in the milk extender (47 vs. 46.5%; NS), as was membrane integrity (38 vs. 37.7%; NS) and the percentage of uncapacitated spermatozoa (28.5 vs. 26.3%; NS). There were no statistically significant differences in fertility rates found between Bioexcell and the control extender: NRR-21 (35.9 vs. 33.2%), NRR-36 (34.8 vs. 32.6%), and PR-US (28.4 vs. 27.2%). In conclusion, Bioexcell appears to be an alternative to the conventional milk-egg yolk extender for freezing ram semen, and provides similar fertility results after cervical AI under extensive management conditions. Thus, Bioexcell, containing no additives of animal origin, can offer a safer alternative when frozen semen is used for introducing new genetic material into a flock or a country.