Efecto de etilenglicol y leche en polvo en la criopreservación de semen de bocachico Prochilodus magdalenae / Effect of ethylene glycol and milk powder in the cryopreservation of bocachico semen Prochilodus magdalenae

RESUMEN Bocachico Prochilodus magdalenae es una especie endémica y la más importante de la pesquería continental colombiana. No obstante, sus capturas han disminuido aproximadamente el 67% en los últimos cuarenta años, por tanto ha sido categorizada como vulnerable a la extinción. La criopreservación de semen, es una herramienta biotecnológica de conservación por tanto el objetivo del presente estudio fue evaluar la criopreservación de semen de bocachico con etilenglicol (EG) y leche en polvo descremada (LP). La solución crioprotectora estuvo compuesta por EG (6, 8 o 10%), LP (3, 5 o 7%) y glucosa 6%. La calidad del semen descongelado se evaluó con un software tipo CASA (computer assisted semen analysis). El porcentaje de inclusión de EG, no afectó significativamente ninguno de los parámetros de calidad seminal evaluados (p>0,05), a excepción de la tasa de eclosión (p<0,05); mientras que, la LP afectó significativamente el porcentaje de espermatozoides estáticos (p<0,05) y las tasas de fertilización y eclosión (p<0,01). La mayor movilidad total se obtuvo cuando EG se incluyó a 10% y la LP a 7% (38,4±18,4%) (p<0,05); pero las mayores tasas de fertilización (54,3-64,2%) y eclosión (47,7-57,5%) se obtuvieron cuando EG se incluyó a 6 u 8% y la LP se incluyó a la menor concentración evaluada (3%), sin observarse diferencia significativa entre estos tratamientos (p>0,05). Los resultados permiten concluir que la combinación EG 6% con LP 3% permiten la criopreservación de semen de Prochilodus magdalenae de buena calidad y capacidad fecundante.

ABSTRACT Bocachico Prochilodus magdalenae is an endemic species and the most important of the Colombian continental fishery. Its catches have decreased by approximately 67% in the last forty years and, it has been categorized as extinction vulnerable. Semen cryopreservation is a biotechnological conservation tool; therefore, the aim of this study was to evaluate bocachico semen's cryopreservation with ethylene glycol (EG) and skimmed milk powder (LP). The cryoprotective solution was composed of EG (6, 8 or 10%), LP (3, 5 or 7%) and glucose at 6%. The quality of the thawed semen was evaluated with CASA software (computer assisted semen analysis). The inclusion percentage of EG did not significantly affect any of the evaluated semen quality parameters (p>0,05), except for the hatching rate (p <0.05). In contrast, LP presented significant effects on the percentage of static sperm (p <0,05) and on fertilization and hatching rates (p<0,01). The highest total motility was achieved with EG included at 10% and the LP 7% (38,4±18,4%) (p<0,05); but the highest fertility rates (54,3-64,2%) and hatching (47,7-57,5%) were registered when EG included at 6 or 8% and LP included at the lowest rate evaluated (3%), no significant difference was observed between these treatments (p>0,05). The results allow us to conclude that the combination EG 6% with LP 3% allows the cryopreservation of Prochilodus magdalenae semen of good quality and fertilizing capacity.

Influencia de agentes crioprotectores sobre las cinéticas de congelación de lechuga (Lactuca sativa L.) / Influence of cryoprotective agents on lettuce freezing kinetics (Lactuca sativa L.)

RESUMEN La lechuga es una hortaliza de hoja que no resiste la congelación. Como una alternativa de conservación de lechuga Lactuca sativa L. "Lollo Bionda", se evaluó el efecto de la aplicación de crioprotectores sobre cada una de las etapas de congelación de lechuga. En cada etapa, se evaluó el tiempo de duración, la velocidad y la temperatura de congelación. Como crioprotectores, se utilizaron soluciones de Aloe (Aloe barbadensis Miller), a concentraciones de 70, 80 y 90%; almidón, a concentraciones de 0,5, 1 y 2% p/p y aceite de oliva. Las muestras, se impregnaron con los diferentes crioprotectores y se ultracongelaron. Las temperaturas del material vegetal, se registraron durante una hora, con intervalos de 5s; se construyeron las cinéticas de congelación y sobre las cinéticas, se identificaron las diferentes etapas de congelación. Se encontró que el tipo y la concentración del crioprotector altera, de manera estadísticamente significativa, la temperatura, el tiempo y la velocidad de cada una de las etapas de congelación. Las mayores concentraciones de solutos presentes en Aloe vera al 90%, almidón al 2% y aceite de oliva influyeron considerablemente en la acción crioprotectora de la lechuga.

ABSTRACT Lettuce is a leafy vegetable that does not resist freezing. As an alternative to the lettuce Lactuca sativa L. "Lollo Bionda" lettuce conservation, the effect of the application of cryoprotectants on each of the freezing stages of lettuce was evaluated. In each stage, duration time, speed and freezing temperature were evaluated. Aloe (Aloe barbadensis Miller) solutions at concentrations of 70, 80 and 90%, starch at concentrations of 0.5, 1 and 2% w / w and olive oil were used as cryoprotectants. The samples were impregnated with the different cryoprotectants and deep-frozen. The temperatures of the plant material were recorded for one hour at intervals of 5 s, the freezing kinetics were built and, on the kinetics, the different stages of freezing were identified. It was found that the type and concentration of the cryoprotectant alters in a statistically significant way the temperature, time and speed of each of the freezing stages. The highest concentrations of solutes present in 90% Aloe vera, 2% starch and olive oil significantly influenced the cryoprotective action of lettuce.

Cryopreservation of banana's cv Grand Naine in vitro rhizomes

ABSTRACT The preservation of banana genetic material is usually performed through seedlings. However, most banana cultivars do not produce seed and are propagated vegetatively. Therefore, cryopreservation is a feasible technique that allows the preservation of banana genotypes indefinitely. For the success of cryopreservation protocols, the selection of cryoprotectants and pre-freezing techniques are important factor. Therefore, the objective of this study was to verify the effects of different cryoprotectants with and without 1% phloroglucinol and pre-cooling periods on the development of a protocol for cryopreservation of in vitro rhizomes ofMusa accuminata(AAA) cv Grand Naine banana. The addition of 1% phloroglucinol to the cryoprotective solutions, such as PVS2 enhanced recovery of cryopreserved banana rhizomes. In addition, pre-cooling of explants in ice for 3 hours in PVS2 + 1% of phloroglucinol allowed efficient cryopreservation of banana rhizomes, followed by successful recovery and regeneration of in vitro shoots of banana cv Grand Naine.

Lyophilization (Drying Method) Cause Serious Damages to the Cell Viability of Lactic Acid Bacteria

Preservation of industrial’s lactic acid bacteria (probiotics) by freeze-drying. Lactic acid bacteria have important nutritional needs and do not have resistance against the environmental conditions surrounding their production (drying, storage, etc.) and their use in vivo (physico-chemical properties of the digestive tract). In this condition, industrials and microbiologists develop regularly research projects of new lactic bacteria able to support the whole of the processes of production, storage and formulation without losing their functional properties. Among various methods of drying (atomization, fluidization and freeze-drying), freeze-drying makes it possible to obtain a thorough dehydration compatible with very long storage times. This method involves changes in product temperature and cause damage to microorganisms because it requires freezing that is not without consequences for cells. On the other side, it causes cellular (peroxydation of the fatty-acids) and genetic (proteins’s modifications) deteriorations. Using cryoprotectants and antioxidants during freeze-drying storage increases appreciably the rate of viability of these cells.

Effect of cryoprotectant removal by microfluidic chip on developmental capacity of oocytes / 生物医学工程学杂志

In order to reduce osmotic damage and chemical toxicity of cryoprotectants (CPA) to oocytes during unloading process, the microfluidic chip was used to remove CPA from porcine MⅡ oocytes in this study. Firstly, the effects of unloading time, composition and concentration of diluting solutions of microfluidic method on survival rate and developmental capacity of oocytes were studied, then microfluidic method was compared with traditional one-step and two-step CPA unloading protocols. The results showed that when the total time is 8 minutes, the survival rate and morula rate of oocytes treated with microfluidic method could achieve 95.99% ± 4.64% and 74.17% ± 1.18%, respectively, which were not significantly different from fresh control group (98.53% ± 2.94%; 78.22% ± 1.34%). In addition, 1 mol/L sucrose diluting solutions were more beneficial than other solutions, and it was also showed that microfluidic method achieved better survival, cleavage rate of oocytes than traditional methods. Microfluidic CPA removal protocol can reduce the damage to oocytes during unloading process, and may further improve the cryopreservation effect of oocytes.

Cryopreservation of testicular tissue: an alternative to maintain the reproductive capacity in different animal species / Criopreservação do tecido testicular: uma alternativa para manter a capacidade reprodutiva em diferentes espécies animais

ABSTRACT: Cryopreservation of testicular tissue enables the maintenance of reproductive capacity in different animal species, and contributes to the formation of gene banks for endangered species. The spermatogonia present in the testes can be grown in vitro and the sperm obtained can be used in artificial breeding programs. This review aimed to describe the main techniques of testicular cryopreservation, the main cryoprotectants used, as well as the progress made in different animal species thus far. In the last decade, significant progress has been made in obtaining viable and functional germ cells from testicular tissue. However, more research is needed to better establish protocols that can be used in clinical practice with various species.

RESUMO: A criopreservação do tecido testicular possibilita a manutenção da capacidade reprodutiva em diferentes espécies animais e contribui para a formação de bancos de germoplasma nas espécies ameaçadas de extinção. As espermatogônias presentes nos testículos podem ser cultivadas in vitro e os espermatozoides obtidos utilizados em programas de reprodução artificial. Assim, esta revisão teve como objetivo descrever as principais técnicas de criopreservação testicular, os principais crioprotetores utilizados e os avanços obtidos até o presente momento nas diferentes espécies animais. Na última década, os avanços obtidos com a utilização do tecido testicular para obtenção de células germinativas viáveis e funcionais foram significativos. Todavia, o estabelecimento de protocolos que possam ser utilizados na rotina clínica em diferentes espécies ainda necessitam de maiores esclarecimentos.

Métodos de conservación para actinobacterias con actividad solubilizadora de fósforo / Preservation methods for actinobacterias with phosphate solubilizing activity

El objetivo de esta investigación fue evaluar diferentes métodos de conservación para actinobacterias solubilizadoras de fósforo debido a la poca información de métodos específicos reportados para estos microorganismos. Los métodos de conservación se evaluaron a 3 diferentes periodos de tiempo; largo, mediano y corto plazo, usando métodos de congelación y liofilización; arcilla, sílica, arena y transferencia periódica, respectivamente. Para ello se usaron 15 aislamientos de 3 localidades diferentes (La Vega, Maní y Tota) y un banco de referencia de la Unidad de Investigaciones Agropecuarias de la Pontificia Universidad Javeriana. Se prepararon todos los inóculos en solución salina al 0,85% (p/v) y se ajustaron a una concentración de 10(8) cel/mL, seguido a ello se inocularon los viales de cada método de conservación con sus respectivos crioprotectantes, glicerol 20% (v/v), 30% (v/v) para congelación y skim milk 18% (p/v) para liofilización. Los métodos a mediano plazo se ejecutaron de igual manera, el inóculo se agregó a 10 perlas de arcilla, 10 g de arena y 5 g de sílica, posteriormente se almacenaron a 4 °C. El método de corto plazo se evaluó en agar avena (15 g/L). La evaluación se realizó mediante recuento directo en cámara de Neubauer por la técnica de azul de tripán, además de la caracterización macroscópica y microscópica de cada aislamiento en transferencia periódica. Se estableció que la actividad solubilizadora de fósforo se mantuvo más estable en los métodos de glicerol 30 % (p/v) y liofilización según el análisis estadístico.

The aim of this research was to evaluate different methods of preservation for actinobacteria with phosphate solubilization activity due to there are a few specific methods reported for these organisms. The methods were evaluated at three different time periods; long, medium and short-term and employing methods as cryopreservation and dry-freezing; clay, silica and sand; and periodically plating, respectively. Therefore 15 isolates from 3 different locations (La Vega, Maní and Tota) and a bank reference from Livestock Research Unit of the Pontificia Universidad Javeriana were used. All inocula were prepared in 0.85% saline solution (w/v) which were adjusted to a concentration of 10(8) cells/mL, followed each vial was inoculated with their respective storage cryoprotectants , glycerol 20% (v/v), 30% (v/v) to freeze and 18% skim milk (w/v) for dry-freezing. Medium-term methods were performed similarly; the inoculum was added to 10 clay beads, 10 g of sand and 5 g of silica and then stored at 4 °C. The short-term method was evaluated in oatmeal agar (15 g/L). The evaluation was performed by direct counting in a Neubauer chamberusing trypan blue staining technique, in addition to macroscopic and microscopic characterization of each isolate in periodic plating. It was established that the phosphorus solubilizing activity was more stable in glycerol 30% (w/v) and lyophilization for statistical analysis.

Efecto de la concentración de crioprotectores sobre la viabilidad post vitrificación de embriones murinos (mus musculus) / Effect of cryoprotectant concentration on the post vitrification morphological viability of murine embryos (mus musculus)

Con la finalidad de determinar la mejor solución de vitrificación (SV), usando Etilenglicol (EG), Glicerol (G) y Sucrosa (SUC), se evaluó la apariencia morfológica post vitrificación de embriones murinos (Mus musculus). Ocho mezclas diferentes de crioprotectores fueron evaluadas, con las siguientes concentraciones: SV1: 0% de crioprotectores; SV2: 50% EG; SV3: 50% G; SV4: 50% SUC; SV5: 25% EG + 25% G; SV6: 50% EG + 0,3M SUC; SV7: 50% G + 0.3M SUC y SV8: 25% EG + 25% G + 0,3M SUC. El mayor número de embriones (94,7%) con apariencia morfológica normal, fueron los equilibrados con SV5. No hubo diferencia significativa entre la SV8 (88,9%) y la SV5. Mientras que los embriones criopreservados con las soluciones restantes, presentaron viabilidad morfológica más baja (p<0,05). Estos resultados sugieren que la SV5 provee mejor tolerancia al proceso de vitrificación, observándose en los embriones la más alta viabilidad y la más baja frecuencia de anormalidades morfológicas. Estos hallazgos contribuyen de manera importante, en la selección de los crioprotectores para la vitrificación.

In order to determine the best vitrification solution (VS), using ethylene glycol (EG), glycerol (G) and sucrose (SUC), the post vitrification morphology in murine embryos (Mus musculus) was evaluated. Eight different mixtures of these chemicals, with the following concentrations: VS1: 0%; VS2: 50% EG; VS3: 50% G; VS4: 50% SUC; VS5: 25% EG + 25% G; VS6: 50% EG + 0.3M SUC; VS7: 50% G + 0.3M SUC and SV8: 25% EG + 25% G + 0.3M SUC, were used. VS5 was the solution with the highest percentage (94,7%) of embryos with normal morphology. There were no significant differences between the VS8 (88.9%) and VS5. However, embryos cryopreserved with the other solutions had a lower morphological viability (p<0.05). These results suggest that VS5 provides embryos with better tolerance to the vitrification process, because a higher viability and lower frequency of morphological abnormalities were present. These findings provide details of great importance to the selection of cryoprotectants for vitrification.

Tiempo de supervivencia in vivo y criopreservación de Trypanosoma vivax / In vivo survival time and cryopreservation of Trypanosoma vivax

Uno de los problemas más comunes del trabajo con Trypanosoma vivax, es la supervivencia y criopreservación de este protozoario, lo cual origina pérdida de aislados de campo y errores en exámenes parasitológicos. Se propone evaluar la supervivencia in vivo en condiciones de campo y criopreservación de T. vivax. Para determinar la supervivencia, la sangre se sometió a temperatura ambiente y refrigeración a 4°C, luego se determinó la sobrevivencia en el tiempo. Para el estudio de criopreservación, se emplearon dos crioprotectores de diferente naturaleza química: glicerol 10 por ciento y DMSO 5 por ciento de concentración final. Además, la criopreservación se realizó bajo tres condiciones de almacenamiento en nitrógeno líquido: 1) fase gaseosa 2) líquida y 3) combinación de ambas. Durante la evaluación de la supervivencia, se observó que la sobrevivencia de T. vivax en sangre refrigerada disminuyó significativamente (P<0,01), en comparación con aquellas sometidas a temperatura ambiente. Sin embargo, la sobrevivencia de éstos últimos comienza a disminuir luego de 6 horas, aunque algunos hemoparásitos permanecieron viables hasta 24 horas post-recolección. Para evaluar la criopreservación, al cabo de dos semanas, se descongelaron los crioviales, se determinó la sobrevivencia, resultando negativas las muestras sometidas a congelamiento directo en fase líquida. Los otros dos métodos empleados, resultaron similares (estadísticamente no significativos), el glicerol 10 por ciento resultó con mayor número de parásitos viables. En conclusión, se determinó que, las muestras infectadas con T. vivax deben evaluarse antes de 8 horas post-recolección y mantenerlas a temperatura ambiente. Por otra parte, el congelamiento debe realizarse en primera instancia en fase gaseosa o combinación gaseosa/líquida, empleando glicerol 10 por ciento. Estos resultados, permiten sugerir la mejor metodología a ser empleada para la supervivencia de los parásitos antes de exámenes parasitológicos...

One of the common problems working with Trypanosoma vivax is its survival and cryopreservation, which originates loss of field isolates and parasitological examinations mistakes. The aim of this paper was to study the best methodologies for in vivo survival under field conditions and cryopreservation of the T. vivax. In order to study complete blood survival of T. vivax, two surviving conditions were tested at: room temperature and refrigeration at 4°C. The result shows that surviving in cooled sampled diminished significantly (P<0.01) compare with room temperature. Nevertheless, surviving of room temperature parasite begins to diminish after 6 hours, although some parasites remained viable up to 24 hours post-harvesting. Cryopreservation studies were made under three liquid nitrogen storage conditions: 1) gaseous phase 2) liquid and 3) gaseous/ liquid phase combination (glycerol 10 percent and DMSO 5 percent, were used as cryoprotectants). After two weeks and defrost the survive of T. vivax from cryovials determined. The result show that: a) direct freezing in liquid phase samples were negative and b) the other two methodology were positive and statistically similar, glycerol 10 percent resulted with the greatest number of viable parasites. In conclusion, these results suggest that the best methodologies for conservation under field conditions, were that the samples infected with T. vivax must be evaluated before 8 hours post-harvesting at room temperature and cryopreservation condition of the T. vivax, must be made in gaseous phase or gaseous/liquid phase combination.

Criopreservação de espermatozóides eqüinos comparando duas curvas de congelamento combinadas com diluentes comerciais: uma análise laboratorial / Cryopreservation of equine spermatozoa comparing different freezing rates combined with commercial extenders: laboratorial analysis

Durante o processo de criopreservação de sêmen, os espermatozóides sofrem alguns danos que resultam na diminuição da fertilidade deste. O presente estudo foi realizado com o objetivo de avaliar os efeitos da utilização combinada de duas curvas de congelamento com dois diluentes comerciais (FR-5® e Botu-Crio®) sobre a criopreservação de sêmen eqüino. Foram analisados 20 ejaculados de dois garanhões. As amostras foram avaliadas por microscopia de contraste de fase e microscopia de epifluorescência, observando-se a motilidade progressiva e total do sêmen pós-descongelamento e a integridade e a funcionalidade da membrana dos espermatozóides. A combinação entre curva automatizada e Botu-Crio® apresentou as maiores médias nas análises de motilidade total e progressiva, após o descongelamento. O diluente Botu-Crio®, isoladamente, preservou também as membranas destes, quando foram realizadas as análises de integridade utilizando teste com diacetato de carboxifluoresceína e iodeto de propídio e funcionalidade de membrana pelo teste hiposmótico.

During semen cryopreservation, sperm cells were submitted to several deleterious events leading to membrane damage which result in fertility decrease. This study was designed to compare the effects of two freezing techniques (conventional and automated), and the use of two commercial extenders as cryoprotectants (FR-5® and Botu-Crio®) on total and progressive motility, integrity and functionality of spermatic membranes during the cryopreservation of equine semen. Twenty ejaculates from two stallions were analyzed. The total and progressive motility of fresh and post-thawing semen samples were evaluated by patterns assays. Function of plasmatic membrane was measured by the hipoosmotic swelling test. Integrity of plasmatic membrane was evaluated using carboxifluorescein diacatate and iodidium propide fluorescent probes. There were significant differences between the two freezing techniques and/or between cryoprotectants for all assessed parameters. The combination of Botu-Crio® and automated curves showed better results on total and progressive post-thawing motility. The extender Botu-Crio®, alone, showed to better preserve the membrane integrity and function.

Sucesso do resfriamento e congelamento de sêmen de pirapitinga Brycon nattereri / Sucess of cooling and freezing of pirapitinga (Brycon nattereri) semen

Avaliaram-se protocolos de resfriamento e de criopreservação do sêmen de pirapitinga (Brycon nattereri) utilizando-se sêmen diluído em NaCl 154mM, NaCl 200mM, Saad e BTS® e resfriado por sete dias. Cinco diluidores (glicose 277mM, NaCl 154mM, NaCl 200mM, Saad e BTS®) foram combinados com dois crioprotetores (DMSO - dimetilsulfóxido e metilglicol) e usados como meio de congelamento. O sêmen diluído em cada meio foi envasado (palhetas de 0,5ml) e congelado, e a motilidade espermática avaliada após o descongelamento (60ºC, 8seg). O sêmen foi novamente congelado em palhetas com diferentes volumes (0,25 e 0,5ml) e descongelados em banho-maria em duas temperaturas (50º e 60ºC). As maiores motilidades (48 por cento) foram observadas no sêmen diluído em BTS® e resfriado por sete dias. Motilidade espermática acima de 68 por cento foram observadas no sêmen congelado em NaCl 154mM-metilglicol, BTS®-metilglicol, NaCl 200mM-DMSO e Saad-DMSO. Não houve diferença entre os volumes de palheta nem entre as temperaturas de descongelamento quanto a motilidade espermática. Assim, o sêmen de pirapitinga mantém altas taxas de motilidade quando resfriado em BTS® por até sete dias ou congelado em NaCl 154mM-metilglicol, BTS®-metilglicol, NaCl 200mM-DMSO e Saad-DMSO

Cooling and freezing protocols of pirapitinga (Brycon nattereri) semen were evaluated using semen diluted in 154mM NaCl, 200mM NaCl, Saad or BTSÕ, and cooled for seven days. Sperm motility was daily evaluated. Five extenders (277mM glucose, 154mM NaCl, 200mM NaCl, Saad and BTSÕ) were combined with two cryoprotectants (DMSO - dimethyl sulphoxide and methylglycol) to produce 10 cryosolutions. Semen was diluted in each cryosolutions, aspirated into 0.5ml straws and frozen. Sperm motility was evaluated after thawing (60ºC, 8 sec). Then, semen was frozen in straws with different volumes (0.25 and 0.5ml), and thawed under different water-bath temperatures (50º and 60ºC). Higher sperm motility (48 percent) was observed when semen was cooled in BTSÕ for seven days. Post-thawing sperm motility above 68 percent was observed when semen was frozen in 154mM NaCl-methylglycol, BTSÕ-methylglycol, 200mM NaCl-DMSO or Saad-DMSO. There was no difference on sperm motility when semen was frozen in 0.25 or 0.5ml straws and thawed in 50º or 60ºC water-bath. Thus, pirapitinga semen can be successfully cooled in BTSÕ for seven days or frozen in 154 mM NaCl-methylglycol, BTSÕ- methylglycol, 200mM NaCl-DMSO and Saad-DMSO

A histologic study of bone formation according to different cryoprotectants during cryopreservation of bone

PURPOSE: Several cryoprotectants are in use to help the survival of cells during cryopreservation of bone in maxillofacial region. Among them, Me2SO(dimethyl sulfoxide), EG(ethylene glycol), sucrose were used for experimentally created defects with accompanying cryopreserved bone graft in the rabbit model. The aim of this study is to analyze the effect of above mentioned agents on bone formation using histologic and histomorphometrical methods, thus to provide experimental support for clinical application of these agents. MATERIALS AND METHODS: Nine rabbits were used as experimental animals. Surgical defects were created on the distal femoral heads and mesial tibial heads of each animal using trephine drill(5mm diameter and 5mm length). The harvested bones were cryopreserved in -80 degrees C refrigerator for one week. The defects were filled with cryopreserved bone with cryoprotectants as experimental groups and cryopreserved bone without cryoprotectant as control. Then, the animals were sacrificed at 1, 2, and 3 weeks after surgery. With Goldner's modified Masson trichrome staining and semiautomatic image analysis system, we observed the change of the cells and bone formation. RESULTS: After bone graft, bone formation and active remodeling process were examined in all experimental groups and the control. But the intensity of such activities of the control were somewhat weaker than that of the experiments. Especially Me2SO+ sucrose group was the best in bone formation and bone remodeling. Me2SO group was more than that of EG group in bone fomation. Sucrose seems to be helpful in survival of the bone cell. Histologic findings showed superior bony quantity and quality in experimental groups than that in control. CONCLUSIONS: The data from this study provides the basis for future studies for evaluating the effect of cryoprotectants in the cryopreservation of bone and clinical study for predictable use of these agents.

Successful pregnancy derived from slowly freezing donated human oocytes by intracytoplasmic sperm injection / 中华泌尿外科杂志

Objective To set up a novel clinical method for storage of oocytes by slowly freezing and successful pregnancy by intracytoplasmic sperm injection (ICSI). Methods The frozen oocytes (n=33) in metaphase Ⅱ (28),which were normal in morphology,were denuded of their cumulus-corona complex.The protective freezing solution contained 1,2-propanediol (1.5 mol/L) and sucrose (0.35 mol/L) and the process was slow for freezing and rapid for thawing.The recipients of these oocytes received hormone replacement therapy (HRT). Results A high survival rate of oocytes (32/33) was obtained when the sucrose concentration was 0.35 mol/L.ICSI induced a higher fertility rate (23/28),a good embryo cleavage rate (21/23) and a satisfactory embryo morphology for thawed oocytes.Embryo transfers were performed in 6 cycles/6 cases. Four cases got clinical pregnancy by demonstration of four gestation sacs on B ultrasonic examination 7 weeks after ICSI,1 fetal was first trimester spontaneous abortion,another 3 survival fetal were ongoing pregnancy and the first case gestation week is 17 weeks. Conclusions This study is the first successful application of human oocyte cryopreservation in China and this method can increase the survival rate of freezing oocytes.It’s easy to carry out,low in cost and high in the recovery rate of oocytes after thawing.