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1.
Malaysian Journal of Medicine and Health Sciences ; : 125-132, 2022.
Article in English | WPRIM | ID: wpr-980467

ABSTRACT

@#Introduction: The cryopreservation of periodontal ligament stem cells (PDLSCs) required a good combination of CPA composition as a step in the preparation of PDLSCs. This study aimed to analyze the proliferative capacities and differentiation potentials of PDLSCs after slow-freezing cryopreservation with CPA in different combinations. Methods: The fourth passage of the primary PDL cells were examined their fibroblast-like morphology and colony forming unit-fibroblast (CFU-F), and characterized by surface markers for mesenchymal stem cells using flow cytometry. PDLSCs were divided into two groups of freshly-PDLSCs (fPDLSCs) and cryopreserved-PDLSCs (cPDLSCs). The PDLSCs were cryopreserved using slow freezing method with CPA in different combinations: 1) 90%FBS+10%DMEM (FD-group), 2) 90%DMEM+10%DMSO (DDs-group), 3) 90%FBS+10%DMSO (FDs-group), and 4) 100% Cell Banker (CB-group) as positive control. The proliferation of fPDLSCs and cPDLSCs were evaluated by trypan blue dye exclusion method. The multipotency of cells was assessed by Oil Red O, Alizarin Red, and Alcian Blue staining. Results: The primary PDL cells had fibroblast-like morphology and CFU-F ability. They expressed more than 95% positive MSC surface markers of CD90, CD73, CD150, and CD44, but showed less than 2% hematopoietic cell markers of CD11b/CD19/CD34/CD45 and HLA-DR. The cPDLSCs viability of FDs-group was 81.5% and 80% in -80oC and LN2, respectively. The fPDLSCs and cPDLSCs proliferation and doubling time were no statistically significant difference (p>0.05). They could differentiate into adipogenic, osteogenic, and chondrogenic differentiation. Conclusion: The cPDLSCs could maintain their proliferative capacities and differentiation potentials after slow-freezing cryopreservation with 90%FBS+10%DMSO in -80oC.

2.
Chinese Journal of Experimental Traditional Medical Formulae ; (24): 182-188, 2022.
Article in Chinese | WPRIM | ID: wpr-940303

ABSTRACT

ObjectiveTo establish a simple, fast and accurate method for locating the volatile oil in Angelicae Sinensis Radix based on frozen section and fluorescence imaging technology, and to reveal the distribution and accumulation of volatile oil in the roots of this herbal medicine. MethodAngelicae Sinensis Radix was used as the research material, the best frozen section conditions for the research material were established by comparing the effects of different cryoprotectants on the quality of frozen sections of Angelicae Sinensis Radix. The suitability of Sudan Ⅲ chemical staining and fluorescence localization for positioning the volatile oil were compared according to the loss of volatile oil and the complexity of operation process. ResultA new method for evaluating the quality of frozen sections of Angelicae Sinensis Radix was established. According to the evaluation equation, it was found that the highest score was obtained when the head, body and tail positions of Angelicae Sinensis Radix were treated with 20% glycerol, 15% glycerol and 20% sucrose, respectively. There was yellowish-brown oily substance in the oil chambers of phelloderm and secondary phloem, and oil canal of the secondary xylem of Angelicae Sinensis Radix, which could be stained orange red or orange yellow by Sudan Ⅲ, and there was green spontaneous fluorescence in the same part under the fluorescence microscope. ConclusionThe relatively complete section of Angelicae Sinensis Radix can be obtained after being treated with cryoprotectant. The volatile oil exists in the oil chambers of phelloderm and secondary phloem, and oil canal of the secondary xylem of Angelicae Sinensis Radix. This study can provide reference for observation of the accumulation sites of volatile oil in other plants.

3.
Rev. colomb. cienc. pecu ; 34(3): 200-211, July-Sept. 2021. tab, graf
Article in English | LILACS-Express | LILACS | ID: biblio-1408021

ABSTRACT

Abstract Background: In artificial insemination, chicken egg yolk is added to bovine semen to protect it during the cryopreservation process, although it contains substances that can affect the microbiological quality and metabolism of sperm. Objective: To evaluate post-thaw quality of bovine cryopreserved semen added with centrifuged and non-centrifuged egg yolk, low-density lipoproteins (LDL), and trehalose (T). Methods: Ten ejaculates from five bulls were cryopreserved under the treatments T1: pure egg yolk (PEY) at 20% v/v, T2: centrifuged egg yolk (CEY) at 20% v/v, T3: LDL at 8% v/v, T4: T at 100 mM, and T5: T at 100 mM plus LDL at 8% v/v (TLDL). Spermatic motility and kinetics, functional membrane integrity (FMI), structural membrane integrity (SMI), sperm vitality (SV) and abnormal morphology (AM) were assessed using the Sperm Class Analyzer (SCA®) system, hypoosmotic test (HOST), SYBR/PI probes, and eosin-nigrosin staining, respectively. A completely randomized design was used. Normal distribution of the variables was validated through the Kolmogórov- Smirnov test. A generalized linear model was used to determine sources of variation. Means were compared using the Tukey test. Results: Inclusion of CEY or LDL had a similar effect on sperm protection, and were superior for motility, kinetics and membrane integrity compared to the other treatments (p<0.05). CEY was superior for progressive motility (p<0.05). The cryoprotective action of LDL was similar to TLDL for motility and kinetics, SMI, SV, and AM (p<0.05). Inclusion of PEY and T resulted in the lowest semen quality (p<0.05). The use of T resulted in a reduction in FMI and SMI (p<0.05). No differences in AM between treatments were found (p>0.05). Conclusions: Egg yolk can be replaced by centrifuged egg yolk or low-density lipoproteins in the freezing extender used for bovine semen used in artificial insemination.


Resumen Antecedentes: la yema de huevo de gallina se agrega al semen bovino usado en inseminación artificial para protegerlo durante el proceso de criopreservación, aunque ésta tiene sustancias que pueden afectar el metabolismo espermatico y la calidad microbiológica del semen. Objetivo: evaluar la calidad post-descongelación del semen bovino criopreservado agregado con yema de huevo centrifugada y no centrifugada, lipoproteínas de baja densidad (LDL) y trehalosa (T). Métodos: diez eyaculados de cinco toros se criopreservaron bajo los tratamientos, T1: yema de huevo pura (PEY) al 20% v/v, T2: yema de huevo centrifugada (CEY) al 20% v/v, T3: LDL al 8% v/v, T4: T a 100 mM, y T5: T a 100 mM más LDL al 8% v/v (TLDL). La movilidad y la cinética espermática, la integridad funcional de la membrana (FMI), la integridad estructural de la membrana (SMI), la vitalidad espermática (SV) y la morfología anormal (AM), se determinaron mediante el sistema Sperm Class Analyzer (SCA®), la prueba hipoosmótica (HOST), las sondas SYBR/PI y la tinción con eosina-nigrosina, respectivamente. Se utilizó un diseño completamente al azar. La normalidad de las variables se validó mediante la prueba de Kolmogórov-Smirnov. Se utilizó un modelo lineal generalizado para determinar las fuentes de variación. Las medias de los tratamientos se compararon mediante la prueba de Tukey. Resultados: CEY y LDL tuvieron un efecto similar en la protección de los espermatozoides, siendo superiores a los demás tratamientos respecto a movilidad, cinética e integridad de la membrana (p<0,05). CEY fue superior para la movilidad progresiva (p<0,05). La acción crioprotectora de LDL fue similar a TLDL para movilidad y cinética, SMI, AM y SV (p<0,05). PEY y T resultaron en la más baja calidad seminal (p<0,05). El uso de T redujo la FMI y la SMI (p<0,05). No se encontraron diferencias en AM entre los tratamientos (p>0,05). Conclusiones: la yema de huevo puede reemplazarse por yema de huevo centrifugada o por lipoproteinas de baja densidad en el diluyente de congelación usado para semen bovino destinado a inseminacion artificial.


Resumo Antecedentes: a gema de ovo de galinha tem sido utilizada com a finalidade de proteger o sêmen bovino durante o processo de criopreservação, embora tenha substâncias que possam afetar o metabolismo dos espermatozóides e a qualidade microbiológica do sêmen utilizado para a inseminação artificial. Objetivo: avaliar a qualidade pós-descongelamento do sêmen bovino criopreservado com gema de ovo centrifugada e não centrifugada, lipoproteínas de baixa densidade (LDL) e trealose (T). Métodos: dez ejaculados de cinco touros foram criopreservados sob os tratamentos, T1: gema de ovo pura (PEY) 20% v/v, T2: gema de ovo centrifugada (CEY) 20% v/v, T3: LDL 8% v/v, T4: T 100 mM e T5: T 100 mM mais LDL 8% v/v (TLDL). Mobilidade e cinética espermática, integridade funcional da membrana (FMI), integridade estrutural da membrana (SMI), vitalidade espermática (SV) e morfologia anormal (AM) foram determinadas por o sistema Sperm Class Analyzer (SCA®), teste hiposmótico (HOST), coloração com SYBR/PI e eosina-nigrosina, respectivamente. Um design completamente aleatoriedade foi usado. A normalidade das variáveis foi validada pelo teste de Kolmogorov-Smirnov. Um modelo linear generalizado foi utilizado para determinar as fontes de variação. As médias dos tratamentos foram comparadas pelo teste de Tukey. Resultados: T2 (CEY) e T3 (LDL) tiveram efeito similar na proteção espermática, sendo superior aos demais tratamentos para mobilidade, cinética e integridade da membrana (p<0,05). T2 (CEY) foi superior para mobilidade progressiva (p<0,05). A ação crioprotetora de T3 (LDL) foi semelhante à T5 (TLDL) para motilidade e cinética, SMI, SV e AM (p<0,05). T1 (PEY) e T4 (T) tiveram a menor qualidade seminal (p<0,05). O uso de T4 (T) produziu uma redução na SMI e FMI (p<0,05). Não foram encontradas diferenças na AM entre os tratamentos (p>0,05). Conclusões: a gema de ovo pode ser substituída por gema de ovo centrifugada ou lipoproteínas de baixa densidade no diluente de congelamento de sêmen bovino.

4.
Braz. J. Vet. Res. Anim. Sci. (Online) ; 58: e168702, 2021. ilus, tab
Article in English | LILACS, VETINDEX | ID: biblio-1344676

ABSTRACT

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)


Subject(s)
Animals , Cyprinidae/embryology , Cryoprotective Agents/analysis , Semen Analysis/veterinary , Dimethyl Sulfoxide/analysis
5.
Asian Journal of Andrology ; (6): 91-96, 2021.
Article in English | WPRIM | ID: wpr-879718

ABSTRACT

Slow freezing is the most commonly used technique for the cryopreservation of spermatozoa in clinical practice. However, it has been shown to have a negative impact on sperm function and structure. Vitrification as a successful alternative method has been proved to have better protective effects on human embryos, but vitrification of spermatozoa is still subject to low recovery rates. In this study, a modified vitrification method for native spermatozoa was developed. A total of 28 semen samples were included; each sample was divided into three equal parts and assigned to fresh, slow freezing, and vitrification groups. Sperm vitality, motility, morphology, DNA integrity, and acrosome reaction were assessed for each of the groups. The results showed that vitrification achieves better results for several sperm protection parameters than slow freezing; vitrification achieves a higher recovery rate (P < 0.05), motility (P <0.05), morphology (P <0.05), and curve line velocity (P <0.05) than slow freezing. Furthermore, DNA fragmentation was decreased (P <0.05) and better acrosome protection (P <0.05) was exhibited in the spermatozoa after vitrification. Principal component analysis of all sperm parameters revealed that the vitrification cluster was closer to the fresh cluster, indicating that spermatozoa are better preserved through vitrification. In conclusion, while both slow freezing and vitrification have negative effects on sperm function and structure, the vitrification protocol described here had a relatively better recovery rate (65.8%) and showed improved preservation of several sperm quality parameters compared with slow freezing.

6.
Chinese Journal of Blood Transfusion ; (12): 926-930, 2021.
Article in Chinese | WPRIM | ID: wpr-1004449

ABSTRACT

Platelet transfusion is one of the important therapeutic methods for clinical prevention of bleeding and hemostasis. At present, agitated storage at (22±2)℃used for platelets leads to risk of bacterial growth, which limits platelet shelf life and safety in transfusion. Cold storage at 4 ℃ can significantly promote the abovementioned drawbacks and present a better hemostatic effect. Platelets storage lesion(PSL) due to the cold storage, however, may lead to platelet activation following transfusion, thus reducing the effective survival rate in vivo. This paper reviews the research advances in PSL and the application of platelets stored at 4 ℃.

7.
Arq. bras. med. vet. zootec. (Online) ; 72(1): 253-262, Jan.-Feb. 2020. tab, ilus
Article in Portuguese | LILACS, VETINDEX | ID: biblio-1088914

ABSTRACT

Os objetivos do presente estudo foram analisar a ultraestrutura do espermatozoide do jundiá amazônico e avaliar a sua criopreservação com três agentes crioprotetores (metanol 10%, DMSO 10% e etilenoglicol 10%) e duas soluções ativadoras (NaCl 0,29% e NaHCO3 1%). Como diluente, foi utilizada uma solução de glicose a 5%, sendo o sêmen envasado em palhetas de 0,25mL e congelado em vapor de nitrogênio (botijão dry shipper). No sêmen fresco, o espermatozoide apresentou comprimento de 25,46±2,54µm, cabeça esférica (1,51±0,18µm), ausência de acrossoma, peça intermediária com formato cônico (0,93±0,17µm), ligeiramente assimétrica, com presença de vesículas, e flagelo único (21,48±2,45µm). O sêmen descongelado apresentou valores mais altos (P<0,05) para duração, vigor e taxa de motilidade espermática com os crioprotetores metanol 10% e DMSO 10%. A duração da motilidade espermática foi maior (P<0,05) com o ativador NaHCO3 1% (21-96 s). O sêmen de Leiarius marmoratus criopreservado com DMSO e metanol apresentou, respectivamente, 7,32±4,21% e 8,94±6,69% de taxa de motilidade. No entanto, os resultados não foram satisfatórios para estabelecer um protocolo para a espécie.(AU)


The aims of this study were to describe the spermatozoon ultrastructure and to evaluate the sperm cryopreservation of the amazon catfish with three cryoprotectant agents (10% methanol, 10% DMSO, and 10% ethylene glycol) and two activator agents (0.29% NaCl and 1% NaHCO3). Glucose 5% extender was used as a diluent solution and sperm loaded in 0.25 straws was frozen in nitrogen vapor (dry shipper). Fresh spermatozoon was 25.46±2.54µm long, the head was spherical (1.51±0.18µm) with no acrosome, the midpiece was cone shaped (0.93±0.17µm) with presence of vesicles, slightly asymmetric, and the flagellum was single (21.48±2.45µm). Post-thawed semen presented higher values (P< 0.05) for duration, vigor and sperm motility rate with cryoprotectants 10% methanol and 10% DMSO. The duration of sperm motility was longer (P< 0,05) when triggered in 1% NaHCO3 (96-21 s). Leiarius marmoratus semen cryopreserved with DMSO and methanol, presented respectively 7.32±4.21% and 8.94±6.69% of motility. However, the results were not satisfactory to establish a protocol for the specie.(AU)


Subject(s)
Animals , Male , Semen Preservation , Spermatozoa/ultrastructure , Catfishes , Cryoprotective Agents
8.
Journal of Biomedical Engineering ; (6): 986-993, 2019.
Article in Chinese | WPRIM | ID: wpr-781837

ABSTRACT

Dimethyl sulfoxide (Me SO) supplemented with fetal bovine serum (FBS) is a widely used cryoprotectant combination. However, high concentration of Me SO is toxic to cells, and FBS presents problems related to diseases such as bovine spongiform encephalopathy and viral infections. Silk protein is a kind of natural macromolecule fiber protein with good biocompatibility and hydrophilicity. The aim of this paper is to analyze the cryoprotective mechanism of silk protein as cryoprotectant. Firstly, differential scanning calorimetry (DSC) was used to measure the thermal hysteresis activity (THA) of silk protein. The THA of 10 mg/mL sericin protein was 0.96°C, and the THA of 10% (V/V) fibroin protein was 1.15°C. Then the ice recrystallization inhibition (IRI) of silk protein-PBS solution was observed with cryomicroscope. The cold stage was set at - 7°C, after 40 minutes' incubation, the mean grain size rate (MGSR) of sericin protein and fibroin protein were 28.99% and 3.18%, respectively, which were calculated relative to phosphate buffer saline (PBS) control. It is indicated that sericin and silk fibroin have certain effects of inhibiting recrystallization of ice crystals. Finally, the structure and physicochemical properties of silk protein were analyzed by Fourier transform infrared spectroscopy (FTIR). The results showed that the content of the random coil was 75.62% and the β-sheet structure was 24.38% in the secondary of sericin protein. The content of the β-sheet structure was 56.68%, followed by random coil structure 22.38%, and α-helix 16.84% in the secondary of fibroin protein. The above analysis demonstrates the feasibility of silk fibroin as a cryoprotectant, and provides a new idea for the selection of cryoprotectants in the future.


Subject(s)
Animals , Bombyx , Calorimetry, Differential Scanning , Fibroins , Sericins , Silk , Spectroscopy, Fourier Transform Infrared
9.
Braz. j. microbiol ; 49(2): 220-231, Apr.-June 2018. tab, graf
Article in English | LILACS | ID: biblio-889224

ABSTRACT

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.


Subject(s)
Basidiomycota/physiology , Cryopreservation/methods , Microbial Viability/radiation effects , Basidiomycota/radiation effects , Cryoprotective Agents/metabolism , Culture Media/chemistry , Time Factors
10.
Pesqui. vet. bras ; 38(2): 350-356, fev. 2018. tab, graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-895565

ABSTRACT

The cryopreservation of somatic tissue in collared peccaries promotes an alternative source of genetic material of this specie. The solid-surface vitrification (SSV) is a great option for tissue conservation; nevertheless, the optimization of SSV requirements is necessary, especially when referred to cryoprotectants that will compose the vitrification solution. Therefore, the aim was to evaluate the effect of the presence of 0.25 M sucrose in addition to different combinations (only or association) and concentrations (1.5 M or 3.0 M) of ethylene glycol (EG) and/or dimethyl sulfoxide (DMSO) in the somatic tissue vitrification of collared peccaries. Subsequently, we tested six combinations of cryoprotectants with or without sucrose in Dulbecco modified Eagle medium (DMEM) plus 10% fetal bovine serum (FBS). Thus, 3.0 M EG with sucrose was able to maintain normal tissue characteristics compared with non-vitrified (control), especially for the volumetric ratio of epidermis (61.2 vs. 58.7%) and dermis (34.5 vs. 36.6%), number of fibroblast (90.3 vs. 127.0), argyrophilic nucleolar organizer region (AgNOR) ratio (0.09 vs. 0.17%) and nucleus area (15.4 vs. 14.5 µm2) respectively. In conclusion, 3.0 M EG with 0.25 M sucrose and 10% FBS resulted in a better cryoprotectant composition in the SSV for somatic tissue of collared peccaries.(AU)


A criopreservação de tecido somático em catetos promove uma fonte alternativa de material genético nesta espécie. A vitrificação em superfície sólida (VSS) é uma ótima opção para a conservação do tecido; contudo, a otimização dos requerimentos da VSS é necessária, especialmente quanto aos crioprotetores que irão compor a solução de vitrificação. Portanto, o objetivo foi avaliar o efeito da presença de 0,25 M de sacarose em adição com diferentes combinações (individual ou associação) e concentrações (1,5 M ou 3,0 M) de etilenoglicol (EG) e/ou dimetilsulfóxido (DMSO) na vitrificação de tecido somático de catetos. Subsequentemente, nós testamos seis combinações de crioprotetores com ou sem sacarose em meio de Eagle modificado por Dulbecco (DMEM) acrescido de 10% de soro fetal bovino (SFB). Assim, 3,0 M de EG com sacarose foi capaz de manter as características normais do tecido comparado com o não vitrificado (controle), especialmente para a proporção volumétrica da epiderme (61,2 vs. 58,7%) e derme (34,5 vs. 36,6%), número de fibroblastos (90,3 vs. 127,0), razão da região argirófila organizadora de nucléolo (AgNOR) (0,09 vs. 0,17%) e área do núcleo (15,4vs.14,5 µm2), respectivamente. Em conclusão, 3,0 M de EG com 0,25 M de sacarose e 10% de SFB resultaram na melhor composição de crioprotetores na VSS para tecido somático de catetos.(AU)


Subject(s)
Animals , Artiodactyla , Cryoprotective Agents , Ethylene Glycol , Sucrose , Tissues/cytology , Vitrification
11.
Rev. colomb. biotecnol ; 19(2): 87-94, jul.-dic. 2017. tab
Article in Spanish | LILACS | ID: biblio-900440

ABSTRACT

RESUMEN El objetivo fue evaluar la calidad del semen descongelado de dorada Brycon moorei crioconservado con dimetilsulfóxido (DMSO) a tres porcentajes de inclusión. El semen se obtuvo de nueve machos mantenidos en cautiverio en la Estación Piscícola Repelón (Atlántico, Col), inducidos con extracto pituitario de carpa (4,5 mg/kg). El semen fue diluido en proporción 1:3 con un diluyente compuesto de DMSO a tres porcentajes 5%, 10% y 15%; glucosa al 6% y yema de huevo al 12%; empacado en macrotubos de 2,5 ml, congelados en vapores de nitrógeno y después de tres meses descongelados a 35°C durante 90 s. Semen fresco fue considerando como tratamiento control. En semen descongelado se evaluó movilidad total, tipos de movilidades, progresividad, velocidades y concentración espermática con el programa Sperm Class Analyzer SCA®; adicionalmente en semen fresco se determinó volumen, color y tiempo de activación. El semen fresco presentó movilidad mayor a 80% y tiempo de activación entre 28,5 y 41 s; mientras que, la concentración espermática osciló entre 10188,1 y 14590,2 millones/ml. La movilidad total del semen descongelado fue mayor cuando DMSO se incluyó a 5% (40,1±5,0%) o 10% (43,3±8,7%) (p>0,05); pero a 15% registró la menor movilidad (30,6±7,9%) y el mayor porcentaje de espermatozoides inmóviles (69.4±7.9%) (p<0,05); lo cual sugiere que inclusiones de DMSO por encima de 10% ocasionan mayores daños al espermatozoide de dorada. Los resultados permiten concluir que DMSO debe ser incluido entre 5 y 10%, junto con glucosa al 6% y yema de huevo al 12% para crioconservar semen de dorada.


ABSTRACT The aim was assess thawed sperm quality of dorada Brycon moorei, cryopreserved with dimethylsulfoxide (DMSO) to three inclusion rate. The sperm was obtained from nine males, kept in captivity in the Repelón Fish Farming Station (Atlántico, Col), were induced with carp pituitary extract (4.5 mg/kg). The semen was diluted with an extender composed of DMSO to three inclusion rates (5%, 10% and 15%); 6% glucose and 12% egg yolk. The sperm was diluted in 1:3, packed in macrotubes of 2.5 mL and freeze with vapors of nitrogen and after three months were thawed at 35°C for 90 s. The fresh sperm was considered as control treatment. The thawed semen was analyzed total motility, types of motility, progressivity, velocities and sperm concentration with the Sperm Class Analyzer SCA® software; further, volume, color and activation time were measured in fresh semen. The fresh sperm showed motility greater than 80% and activation time between 28.5 and 41 s; whereas that sperm concentration ranged between 10188.1 and 14590.2 million/ml. The total motility of thawed sperm was higher when DMSO was included at 5% (40.1±5.0%) or DMSO 10% (43.3±8.7%) (p> 0.05); but with 15% DMSO, were registered the low motility (30.6±7.9%) and the highest percentage of immotile sperm (69.4±7.9%) (p<0.05); which suggests inclusions of DMSO above 10% cause greater damage to dorada spermatozoa. The results showed that DMSO should be included between 5 and 10%, along with 6% glucose and 12% egg yolk for cryopreservation of dorada sperm.

12.
Chinese Journal of Biotechnology ; (12): 817-827, 2017.
Article in Chinese | WPRIM | ID: wpr-242226

ABSTRACT

A rapid quantitative evaluation method for Siraitia grosvenorii cells was successfully developed based on plant cells' capacitance value detected by a viable cell mass monitor and the cryopreservation of S. grosvenorii suspension cells was optimized. The survival rate of S. grosvenorii cells was quantitatively measured by viable cell mass monitor and 2, 3, 5-triphenyltetrazolium chloride (TTC). An optimum cryoprotectant recipe is that the growth medium contained 10% sucrose and 10% DMSO. The experimental results also showed higher cell survival rates and cell viabilities were achieved when suspension cells were treated with pretreatment of 0.2 mol/L sucrose. With the increase of concentration of sucrose, however, the cell survival rate was decreased. And the cell survival rate represented a bell shape with the increase of pretreatment time. The highest cell survival rate and cell viability were obtained with the 9 h' s pretreatment. In addition, there was a good correlation between the cell survival rate measured by cell recovery test and that measured by viable cell mass monitor, while there were no significant differences in the cell morphology and the ability of mogrosides V production by S. grosvenorii cells cultured in suspension after cryopreservation. Therefore, the evaluation method developed based on the viable cell mass monitor has good feasibility and reliability.

13.
Chinese Traditional and Herbal Drugs ; (24): 1314-1320, 2017.
Article in Chinese | WPRIM | ID: wpr-852871

ABSTRACT

Objective: To prepare silybin (SLB) proliposomes and evaluate its quality. Methods: SLB proliposomes were prepared by freeze-drying method, and the formulation and process were optimized by single factor investigation and orthogonal design with the encapsulation efficiency and drug loading as indexes. The optimal cryoprotetant was selected and the morphology, particle size, encapsulation efficiency, and stability of SLB proliposomes were investigated. Results: The optimized preparation process was as follows: The ratio of drug to total lipid was 1:12, the ratio of phospholipid to cholesterol was 4:1, the pH of hydration medium was 7.4 and the temperature was 45℃. Mannitol was the optimal cryprotectant to prepare SLB proliposomes, and the formation of SLB proliposomes looked plumpy and compact. The size of preliposome was around (251.40 ± 2.14) nm, the Zeta potential was around (-30.80 ± 0.89) mV, encapsulation efficiency was (88.92 ± 5.86)%, and it had good stability during storage. Conclusion: The preparation process of SLB proliposomes is simple, and it has high encapsulation efficiency and good stability, therefore, it is deserved to be further studied.

14.
Pesqui. vet. bras ; 36(3): 209-215, mar. 2016. tab, graf
Article in English | LILACS | ID: lil-782058

ABSTRACT

The objective of this study was to evaluate the vitrification of bovine preantral follicles with dimethylsulfoxide (D) and sucrose (S) plus α-tocopherol 5mmol/L (T5) or 10mmol/L (T10) and, evaluate the thawed with minimal essential medium (m) with or without sucrose (s). Ovaries of cows were collected from slaughterhouse for the experiment I (n=66) and II (n=51). In the laboratory ovarian fragments were randomly assigned either to fresh control and 8 vitrification treatments (Controle and Dm; Dms, DSm; DSms; DST5m; DST5ms; DST10m; DST10ms). Ovarian fragments were placed in vitrification solution (5 min) and immersed in liquid nitrogen (-196°C), after a week, the fragments were thawed and analyzed. In the experiments I, preantral follicles were morphologically observed for histological evaluation, (normal; degenerated and developing of stage). In the experiment II, preantral follicles were mechanically isolated from ovarian tissue and examined with trypan blue, where dead and live corresponded to stained or non-stained. The treatments DSm, DSms and DST10m were effective in preserving the morphology in situ. However, the viability of isolated preantral follicles after vitrification remained high only in treatment DST10m. Thus, DST10m preserves survival rates and morphological integrity during vitrification of bovine preantral follicles.


Os objetivos deste estudo foram avaliar a vitrificação de folículos pré-antrais bovinos com dimetilsulfóxido (D) e sacarose (S) adicionando α-tocoferol 5mmol/L (T5) ou 10mmol/L (T10) e, avaliar o aquecimento com meio essencial mínimo (m) com ou sem sacarose (s). Ovários de fêmeas bovinas foram coletados de abatedouro, para o experimento I (n= 66) e II (n= 51). No laboratório fragmentos ovarianos foram distribuídos aleatoriamente para o controle fresco e 8 tratamentos de vitrificação (Controle e Dm; Dms, a DSm; DSms; DST5m; DST5ms; DST10m; DST10ms). Os fragmentos ovarianos foram colocados na solução de vitrificação (5 min) e imersos em nitrogênio líquido (-196°C). Após uma semana os fragmentos foram aquecidos e analisados. No experimento I, folículos pré-antrais foram observados morfologicamente para avaliação histológica (normal, degenerados e estádio de desenvolvimento). No experimento II, folículos pré-antrais foram mecanicamente isolados do tecido ovariano e examinados com o azul de trypan, observando mortos e vivos corados e não corados respetivamente. Os tratamentos a DSm, DSms e DST10m foram eficazes na preservação da morfologia in situ. No entanto, a viabilidade de folículos pré-antrais isolados após a vitrificação manteve-se elevada apenas no tratamento DST10m. Assim, DST10m preservou as taxas de sobrevivência e integridade morfológica durante a vitrificação de folículos pré-antrais bovinos.


Subject(s)
Animals , Female , Cattle , alpha-Tocopherol , Dimethyl Sulfoxide , Ovarian Follicle/anatomy & histology , Oocytes , Sucrose , Vitrification , Antioxidants , Cryopreservation/veterinary , Granulosa Cells
15.
Journal of Huazhong University of Science and Technology (Medical Sciences) ; (6): 153-161, 2016.
Article in English | WPRIM | ID: wpr-285294

ABSTRACT

Organ transplantation is an effective approach for the treatment of end-stage organ failures. Currently, the donor organs used for clinical transplantation are all preserved at above-zero temperatures. These preservation methods are well-established and simple but the storage time lasts for only 4-12 h. Some researchers tried to extend the organ storage time by improving protectant and HLA matching to raise the use of stored organs and prolong the long-term survival of organs. These efforts still fall short of the clinical demand for organ transplantation. Moreover, a great many organs were wasted due to limited storage time, HLA mismatch, patients' conditions or distance involved. Therefore, preserving organs for several weeks or even months and establishing Organ Bank are the tough challenges and have become a shared goal of global scholars. This article reviews some issues involved in the cryopreservation of organs, such as use of cryoprotecting agents, freezing and thawing methods in the cryopreservation of hearts, kidneys and other organs.


Subject(s)
Humans , Cryopreservation , Methods , Cryoprotective Agents , Pharmacology , Organ Preservation , Methods
16.
Braz. j. pharm. sci ; 51(4): 797-802, Oct.-Dec. 2015. graf
Article in English | LILACS | ID: lil-778406

ABSTRACT

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.


Subject(s)
Nanoparticles , Freeze Drying , Ampicillin/pharmacokinetics , Mannitol/therapeutic use , Particle Size , Cryoprotective Agents/analysis
17.
Ciênc. rural ; 45(11): 1939-1945, Nov. 2015. tab, graf
Article in Portuguese | LILACS | ID: lil-762936

ABSTRACT

Genipa americana (jenipapeiro) é uma essência florestal pertencente à família Rubiaceae, que apresenta importância econômica e ambiental, sendo valorizada para produção de alimentos, na recuperação de áreas degradadas, na composição em áreas de preservação permanentes e em sistemas agroflorestais. Objetivou-se avaliar o efeito de diferentes tempos de desidratação em câmara de fluxo laminar e tempos de imersão em solução crioprotetora (MS+0,5M de sacarose) na capacidade regenerativa de ápices caulinares da espécie para estabelecimento de futuros protocolos de criopreservação. Os ápices caulinares foram obtidos de plântulas, acesso Oiteiros, germinadas e cultivadas in vitro. Os explantes foram submetidos ao encapsulamento e a diferentes tempos de imersão em solução crioprotetora e tempos de desidratação em câmara de fluxo laminar. A crioproteção e a desidratação não alteram a viabilidade dos ápices caulinares de jenipapeiro encapsulados ou não encapsulados. A imersão por 24 horas em solução crioprotetora e a desidratação em câmara de fluxo laminar por 2 horas apresentam potencial para uso em futuros trabalhos de criopreservação por encapsulamento-desidratação.


Genipa americana (genipap) is a forest species belonging to the Rubiaceae family that has economic and environmental importance, being valued for food production, recuperation of degraded areas, in the composition in areas of permanent preservation and agroforestry. The objective of this research was to evaluate the effect of different times of dehydration in laminar flow cab and immersion time in cryoprotecting solution (MS+0.5M sucrose) on the regenerative capacity of shoot apices for establishing future cryopreservation protocols. The shoot apices were obtained from seedlings, of Oiteiros accession, germinated and cultured in vitro. Explants were subjected to encapsulation and different exposure times in cryoprotecting solution and dehydration in a laminar flow cab. The cryoprotection and dehydration does not modify the viability of the shoot apices of genipap encapsulated or unencapsulated. Immersion for 24 hours in cryoprotecting solution and the dehydration in a laminar flow cab by 2 hours have potential for use in future studies of cryopreservation by encapsulation-dehydration.

18.
Neotrop. ichthyol ; 13(3): 599-606, July-Sept. 2015. tab, ilus
Article in English | LILACS | ID: lil-760448

ABSTRACT

Cryoprotectant solutions are used to protect the sperm from alterations caused by the low temperature in the cryopreservation process. We evaluated the quality of Colossoma macropomum semen after freezing, using dimethyl sulfoxide (DMSO) as a cryoprotectant, combined with two extender solutions (T1 - Solution 1: Glucose 90.0 g/L, Sodium Citrate 6.0 g/L, EDTA 1.5 g/L, Sodium Bicarbonate 1.5 g/L, Potassium Chloride 0.8 g/L, Gentamycin Sulphate 0.2 g/L, and T2 - Solution 2: Glucose 90.0 g/L, ACP(r)-104 10.0 g/L). Motility rate and motility time did not differ between T1 and T2 and were lower than fresh semen. The number of normal sperm was significantly different in treatments T1 (15.1%) and T2 (21.9%), and both showed a reduction in the percentage of normal sperm compared to fresh semen (57.4%). The values found for the rates of fertilization and hatching, mitochondrial functionality and sperm DNA, did not differ between the treatments (T1 and T2). Regarding membrane integrity, there was a higher percentage of spermatozoa with intact membranes in T1 (53.4%) than T2 (43.7%). The extender solutions, combined with 10% DMSO, maintained the sperm DNA intact in almost all the C. macropomumsperm cells, however there was a loss in their functionality.


As soluções crioprotetoras são utilizadas para proteger os espermatozoides das alterações causadas por baixas temperaturas durante o processo de criopreservação. Avaliamos a qualidade do sêmen de Colossoma macropomumapós o congelamento, utilizando dimetilsulfóxido (DMSO) como crioprotetor, combinado com duas soluções diluidoras (T1 - Solução 1: Glicose 90,0 g/L, Citrato de Sódio 6,0 g/L, EDTA 1,5 g/L, Bicarbonato de Sódio 1,5 g/L, Cloreto de Potássio 0,8 g/L, Sulfato de Gentamicina 0,2 g/L, e T2 - Solução 2: Glicose 90,0 g/L, ACP(r)-104 10,0 g/L). A taxa de motilidade (%) e o tempo de motilidade (s) não diferiram entre T1 e T2, porém foram mais baixos do que no sêmen fresco. O número de espermatozoides normais foi significativamente diferente nos tratamentos T1 (15,1%) e T2 (21,9%), e ambos mostraram uma redução na porcentagem de espermatozoides normais, comparado ao sêmen fresco (57,4%). Os valores encontrados para as taxas de fertilização e eclosão, funcionalidade mitocondrial e DNA do esperma, não diferiram entre os tratamentos (T1 e T2). Para a integridade da membrana, houve uma porcentagem mais elevada de espermatozóides com a membrana intacta em T1 (53,4%) do que T2 (43,7%). As soluções diluentes combinadas com DMSO a 10% preservaram o DNA espermático intacto em quase todas as células do sêmen de C. macropomum, mas houve perda na funcionalidade dos mesmos.


Subject(s)
Animals , Fishes/embryology , Fishes/genetics , Semen Preservation , Semen Preservation/veterinary , Cryoprotective Agents/history
19.
Arq. bras. med. vet. zootec ; 67(3): 945-949, May-Jun/2015. tab
Article in Portuguese | LILACS | ID: lil-779232

ABSTRACT

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.


Subject(s)
Animals , Aloe/embryology , Characiformes , Cryoprotective Agents/analysis , Semen Preservation/veterinary , Cryopreservation/veterinary , Fishes/embryology , Sperm Capacitation , Sperm Motility
20.
China Pharmacy ; (12): 3575-3577, 2015.
Article in Chinese | WPRIM | ID: wpr-501055

ABSTRACT

OBJECTIVE:To optimize freeze-drying technology of albendazole nanosuspension so as to prepare albendazole nanometer powder. METHODS:By adopting freeze-drying method,with particle size and Zeta potential as the indexes,single fac-tor test and verification were made on pre-freezing temperature and the type,ratio and mass fraction of cryoprotectants,and then the albendazole nanosuspension prepared by liquid phase precipitation method was made into albendazole nanometer powder. RE-SULTS:When the pre-freezing temperature was-20℃and the cryoprotectant was 4%glucose-mannitol(3∶7),the average parti-cle size of the prepared nanometer powder was (208.03 ± 2.13) nm,and average Zeta potential was (-15.53 ± 0.18) mV. CON-CLUSIONS:Albendazole nanometer powder with better particle size and potential can be prepared by freeze-drying technology.

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