Somatic cell interspecies nuclear transfer

The low efficiency of the nuclear transfer (NT) procedure requires large number of oocytes to produce embryos and live offspring. A series of experiments were conducted to evaluate the ability of the bovine cytoplast to reprogram nuclei from horses and llamas. In a preliminary study, equine oocytes from small (\u3c20mm diameter) follicles were either pretreated with roscovitine or placed in maturation (IVM only) prior to NT. Roscovitine pretreatment did not improve nuclear maturation rates (roscovitine pretreatment 57% vs. IVM only 66%) and no fusion was obtained from roscovitine-pretreated oocytes after NT. Another preliminary study was conducted with the objective to produce llama NT embryos and to compare their development in two in vitro culture conditions (G1.2® vs. CR1aa). No difference was found in the number of embryos cleaved after 2 d of culture. This resulted in the first scientific report of somatic cell NT, in vitro culture and transfer of NT embryos in the llama. In the next experiment, adult horse and llama fibroblasts were injected into enucleated cow oocytes. The results showed the cow cytoplasm is capable of partially reprogramming nuclei from other species and support mitotic divisions. However, this study also showed a consistent embryonic developmental arrest at the 8- to 16- cell stage when horse or llama donor cells were used as donor nuclei. When a more closely related species of donor cell (banteng) and recipient oocyte (domestic cattle) were used for NT, no embryonic developmental arrest was found. Embryos progressed to achieve high blastocyst rates (banteng male cell line 28% vs. banteng female cell line 15%). Two banteng interspecies NT pregnancies were established and subsequently lost from the banteng male cell line. In the final study, the effect of a mixed mitochondrial population (heteroplasmy) on early embryonic development was investigated. Ooplasmic transfer performed in combination with NT procedure indicated presence of foreign mitochondria clustered in a small portion of the cytoplasm in early stages of embryo development. When goat ooplasm was transferred into interspecies (cow oocyte-goat donor cell) NT embryos, fusion and cleave rates were reduced suggesting an increased level of heteroplasmy or nuclear-ooplasmic incompatibilities

Implications of storage and handling conditions on glass transition and potential devitrification of oocytes and embryos

Devitrification, the process of crystallization of a formerly crystal-free, amorphous glass state, can lead to damage during the warming of cells. The objective of this study was to determine the glass transition temperature of a cryopreservation solution typically used in the vitrification, storage, and warming of mammalian oocytes and embryos using differential scanning calorimetry. A numerical model of the heat transfer process to analyze warming and devitrification thresholds for a common vitrification carrier (open-pulled straw) was conducted. The implications on specimen handling and storage inside the dewar in contact with nitrogen vapor phase at different temperatures were determined. The time required for initiation of devitrification of a vitrified sample was determined by mathematical modeling and compared with measured temperatures in the vapor phase of liquid nitrogen cryogenic dewars. Results indicated the glass transition ranged from -126°C to -121°C, and devitrification was initiated at -109°C. Interestingly, samples entered rubbery state at -121°C and therefore could potentially initiate devitrification above this value, with the consequent damaging effects to cell survival. Devitrification times were calculated considering an initial temperature of material immersed in liquid nitrogen (-196°C), and two temperatures of liquid nitrogen vapors within the dewar (-50°C and -70°C) to which the sample could be exposed for a period of time, either during storage or upon its removal. The mathematical model indicated samples could reach glass transition temperatures and undergo devitrification in 30seconds. Results of the present study indicate storage of vitrified oocytes and embryos in the liquid nitrogen vapor phase (as opposed to completely immersed in liquid nitrogen) poses the potential risk of devitrification. Because of the reduced time-handling period before samples reach critical rubbery and devitrification values, caution should be exercised when handling samples in vapor phase.Fil: Sansinena, Marina Julia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires"; ArgentinaFil: Santos, Maria Victoria. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; ArgentinaFil: Taminelli, Guillermo Luis. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires"; ArgentinaFil: Zaritzky, Noemi Elisabet. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; Argentin

Implications of storage and handling conditions on glass transition and potential devitrification of oocytes and embryos

Devitrification, the process of crystallization of a formerly crystal-free, amorphous glass state, can lead to damage during the warming of cells. The objective of this study was to determine the glass transition temperature of a cryopreservation solution typically used in the vitrification, storage, and warming of mammalian oocytes and embryos using differential scanning calorimetry. A numerical model of the heat transfer process to analyze warming and devitrification thresholds for a common vitrification carrier (open-pulled straw) was conducted. The implications on specimen handling and storage inside the dewar in contact with nitrogen vapor phase at different temperatures were determined. The time required for initiation of devitrification of a vitrified sample was determined by mathematical modeling and compared with measured temperatures in the vapor phase of liquid nitrogen cryogenic dewars. Results indicated the glass transition ranged from -126°C to -121°C, and devitrification was initiated at -109°C. Interestingly, samples entered rubbery state at -121°C and therefore could potentially initiate devitrification above this value, with the consequent damaging effects to cell survival. Devitrification times were calculated considering an initial temperature of material immersed in liquid nitrogen (-196°C), and two temperatures of liquid nitrogen vapors within the dewar (-50°C and -70°C) to which the sample could be exposed for a period of time, either during storage or upon its removal. The mathematical model indicated samples could reach glass transition temperatures and undergo devitrification in 30seconds. Results of the present study indicate storage of vitrified oocytes and embryos in the liquid nitrogen vapor phase (as opposed to completely immersed in liquid nitrogen) poses the potential risk of devitrification. Because of the reduced time-handling period before samples reach critical rubbery and devitrification values, caution should be exercised when handling samples in vapor phase.Fil: Sansinena, Marina Julia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires"; ArgentinaFil: Santos, Maria Victoria. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; ArgentinaFil: Taminelli, Guillermo Luis. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires"; ArgentinaFil: Zaritzky, Noemi Elisabet. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; Argentin

Determination of heat transfer coefficients of biological systems during cooling in liquid nitrogen under film and nucleate pool boiling regimes

The cryopreservation process consists of reducing the temperature of the sample to a point where biological stability is achieved. In particular the measurement of the temperature change of the sample is important to calculate cooling rates and to determine if a sample is vitrified or undergoes phase change transition. As soon an object is plunged into liquid nitrogen it enters into a film boiling regime due to the large temperature difference between the object and the liquid nitrogen (LN2). This determines a heat flux from the object to LN2 causing the latter to boil in the immediate vicinity of the object and creating a pocket of nitrogen vapor around the object which acts as an “insulator” and retards further heat transfer. Film boiling is also referred to as the “Leidenfrost effect”. Boiling curves for a specific cryobiological system are scarcely found in the literature due to the small dimensions of the devices used in the process and the experimental limitations. The experimental information such as the time-temperature curve allows the prediction of the surface heat transfer coefficients that govern the cooling process: film, transition and nucleate boiling. In order to predict the surface heat transfer coefficient for each boiling regime the mathematical modeling of the partial differential equations that represent the energy transfer must be implemented, applying convective boundary conditions. In this work the different heat transfer coefficients and the boiling curve of straws filled with ice (at an initial temperature between -2ºC to -9ºC) were experimentally measured when they were immersed in liquid nitrogen; this allowed to determine the existence of different boiling regimes. The application of a numerical finite element program using the software COMSOL was used to predict time-temperature curves and to obtain the surface heat transfer coefficients that control each boiling regime. Independent experiments were carried out using straws that contained a biological fluid (semen+extender), which were initially at room temperature, to further validate the different surface heat transfer coefficients for film and nucleate pool boiling. The program takes into account the variable thermo-physical properties of the biological sample. This constitutes a highly non-linear mathematical problem, as the freezing process evolves with a variable surface heat transfer coefficients as the different boiling regimes occur. The program was experimentally validated contrasting experimental temperatures vs. time with numerical predictions. The numerical program is an important tool in order to correctly assess the heat transfer process and optimize the cryopreservation of straws filled with biological fluids.Fil: Santos, Maria Victoria. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; ArgentinaFil: Sansinena, Marina Julia. Pontificia Universidad Catolica Argentina "Santa Maria de Los Buenos Aires". Facultad de Ciencias Agrarias; ArgentinaFil: Chirife, Jorge. Pontificia Universidad Catolica Argentina "Santa Maria de Los Buenos Aires". Facultad de Ciencias Agrarias; ArgentinaFil: Zaritzky, Noemi Elisabet. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; Argentin

Determination of heat transfer coefficients of biological systems during cooling in liquid nitrogen under film and nucleate pool boiling regimes

The cryopreservation process consists of reducing the temperature of the sample to a point where biological stability is achieved. In particular the measurement of the temperature change of the sample is important to calculate cooling rates and to determine if a sample is vitrified or undergoes phase change transition. As soon an object is plunged into liquid nitrogen it enters into a film boiling regime due to the large temperature difference between the object and the liquid nitrogen (LN2). This determines a heat flux from the object to LN2 causing the latter to boil in the immediate vicinity of the object and creating a pocket of nitrogen vapor around the object which acts as an “insulator” and retards further heat transfer. Film boiling is also referred to as the “Leidenfrost effect”. Boiling curves for a specific cryobiological system are scarcely found in the literature due to the small dimensions of the devices used in the process and the experimental limitations. The experimental information such as the time-temperature curve allows the prediction of the surface heat transfer coefficients that govern the cooling process: film, transition and nucleate boiling. In order to predict the surface heat transfer coefficient for each boiling regime the mathematical modeling of the partial differential equations that represent the energy transfer must be implemented, applying convective boundary conditions. In this work the different heat transfer coefficients and the boiling curve of straws filled with ice (at an initial temperature between -2ºC to -9ºC) were experimentally measured when they were immersed in liquid nitrogen; this allowed to determine the existence of different boiling regimes. The application of a numerical finite element program using the software COMSOL was used to predict time-temperature curves and to obtain the surface heat transfer coefficients that control each boiling regime. Independent experiments were carried out using straws that contained a biological fluid (semen+extender), which were initially at room temperature, to further validate the different surface heat transfer coefficients for film and nucleate pool boiling. The program takes into account the variable thermo-physical properties of the biological sample. This constitutes a highly non-linear mathematical problem, as the freezing process evolves with a variable surface heat transfer coefficients as the different boiling regimes occur. The program was experimentally validated contrasting experimental temperatures vs. time with numerical predictions. The numerical program is an important tool in order to correctly assess the heat transfer process and optimize the cryopreservation of straws filled with biological fluids.Centro de Investigación y Desarrollo en Criotecnología de Alimento

Experimental determination of surface heat transfer coefficient in a dry ice-ethanol cooling bath using a numerical approach

Background: Dry ice-ethanol bath (-78ºC) have been widely used in low temperature biological research to attain rapid cooling of samples below freezing temperature. The prediction of cooling rates of biological samples immersed in dry ice-ethanol bath is of practical interest in cryopreservation. The cooling rate can be obtained using mathematical models representing the heat conduction equation in transient state. Additionally, at the solid cryogenic-fluid interface, the knowledge of the surface heat transfer coefficient (h) is necessary for the convective boundary condition in order to correctly establish the mathematical problem. Objective: The study was to apply numerical modeling to obtain the surface heat transfer coefficient of a dry ice-ethanol bath. Materials and methods: A numerical finite element solution of heat conduction equation was used to obtain surface heat transfer coefficients from measured temperatures at the center of polytetrafluoroethylene and polymethylmetacrylate cylinders immersed in a dry ice-ethanol cooling bath. The numerical model considered the temperature dependence of thermophysical properties of plastic materials used. Results: A negative linear relationship is observed between cylinder diameter and heat transfer coefficient in the liquid bath, the calculated h values were 308, 135 and 62.5 W/(m2K) for PMMA 1.3, PTFE 2.59 and 3.14 cm in diameter, respectively. Conclusion: The calculated heat transfer coefficients were consistent among several replicates; h in dry ice-ethanol showed an inverse relationship with cylinder diameter.Centro de Investigación y Desarrollo en Criotecnología de Alimento

In-vitro development of vitrified-warmed bovine oocytes after activation may be predicted based on mathematical modelling of cooling and warming rates during vitrification, storage and sample removal

Heat transfer during cooling and warming is difficult to measure in cryo-devices; mathematical modelling is an alternative method that can describe these processes. In this study, we tested the validity of one such model by assessing in-vitro development of vitrified and warmed bovine oocytes after parthenogenetic activation and culture. The viability of oocytes vitrified in four different cryo-devices was assessed. Consistent with modelling predictions, oocytes vitrified using cryo-devices with the highest modelled cooling rates had significantly (P < 0.05) better cleavage and blastocyst formation rates. We then evaluated a two-step sample removal process, in which oocytes were held in nitrogen vapour for 15 s to simulate sample identification during clinical application, before being removed completely and warmed. Oocytes exposed to this procedure showed reduced developmental potential, according to the model, owing to thermodynamic instability and devitrification at relatively low temperatures. These findings suggest that cryo-device selection and handling, including method of removal from nitrogen storage, are critical to survival of vitrified oocytes. Limitations of the study include use of parthenogenetically activated rather than fertilized ova and lack of physical measurement of recrystallization. We suggest mathematical modelling could be used to predict the effect of critical steps in cryopreservation.Centro de Investigación y Desarrollo en Criotecnología de Alimento

Поражение печени у больных инфекционным мононуклеозом

МОНОНУКЛЕОЗ ИНФЕКЦИОННЫЙЛЕЙКОЦИТОВ НАРУШЕНИЯЛИМФОПРОЛИФЕРАТИВНЫЕ НАРУШЕНИЯЭПШТЕЙНА-БАРРА ВИРУСНЫЕ ИНФЕКЦИИПЕЧЕНЬ /ПОВРЕЖ

Buffalo embryos produced by in vitro fertilization from oocytes matured during long-term transport

El búfalo (Bubalus bubalis) es una especie con excelente adaptación a sectores inundables. El mejoramiento genético a través de superovulación y transferencia embrionaria ha tenido escasos resultados debido a difcultades en la detección de celo, pobre respuesta ovárica y limitada recuperación de embriones post-lavaje. La técnica de fertilización in vitro de embriones (FIV) es una biotecnología de gran impacto en el progreso genético. El objetivo del presente trabajo fue estudiar los eventos tempranos de la FIV, analizando la tasa de maduración y desarrollo embrionario post-fertilización de ovocitos madurados in vitro (IVM) durante el transporte. Ovocitos bovinos y bubalinos fueron obtenidos por punción folicular de ovarios post-mortem e IVM durante el transporte por un período de 18 h. Se realizó la FIV con toros de fertilidad comprobada, con una concentración en microgotas de inseminación de 3-4 x 106 espermatozoides motiles/ml por un período de 6 horas. Los embriones fueron cultivados en medio oviductal sintético SOFaa en incubadora gaseada y ambiente humidificado a 38,5ºC durante 9 días. Se evaluaron las tasas de IVM, clivaje (día 2 post-fertilización) y blastocisto (días 7 a 9). Los resultados fueron analizados estadísticamente utilizando Fischer's Exact Test (p<0,05). No se observaron diferencias significativas en la tasa de maduración de ovocitos bubalinos de buena calidad respecto al control sin transporte (72 vs 88%), pero se registró una reducción significativa en la maduración de los ovocitos bubalinos de mala calidad (35%). Asimismo, se lograron producir los primeros embriones bubalinos luego de FIV, aunque las tasas de clivaje (34 vs 70 y 78%) y blastocisto (3 vs 27 y 31%) fueron significativamente menores en búfalos que en bovinos con y sin transporte, respectivamente. Los datos del presente trabajo constituirían el primer informe de FIV en búfalos y producción in vitro de embriones luego de IVM de ovocitos durante el transporte.The water buffalo (Bubalus bubalis) is a species with excellent adaptation to food-prone environments. Genetic improvement using the multiple ovulation and embryo transfer approach has been met with poor results in the buffalo, mainly due to difficulties in heat detection, erratic ovarian response to treatments and low embryo recovery post-flush. In vitro fertilization (IVF) is a powerful reproductive biotechnology that may provide a tool for genetic improvement in this species. The objective of this experiment was to study early embryonic events after IVF in the buffalo, analyzing in vitro maturation and IVF of oocytes matured during ground transportation. Bovine and bubaline oocytes were collected by follicular aspiration of post-mortem ovaries and in vitro matured for 18 h during ground transportation. In vitro fertilization was conducted, semen form bulls of proven fertility was processed and adjusted to a fnal concentration of 3-4 x 106 motile spermatozoa/ml in the insemination drops, oocytes were co-incubated for a period of 6 h. Embryos were then cultured in synthetic oviductal fluid (SOFaa) medium in an incubator and humidified atmosphere at 38.5ºC for 9 days. Oocyte maturation, cleavage and blastocyst rates were evaluated on days 0, 2 and 7 to 9, respectively and results were statistically analyzed using Fischer´s Exact Test (p<0.05). No significant difference was observed in the maturation rate of bubaline oocytes of good quality vs the non-transported control (72 vs 88%); however, the maturation rate of bubaline oocytes of bad quality was significantly lower (35%) than the rest of the groups. Data of present experiment are the first report of buffalo embryos produced by IVF from oocytes matured during transportation, although the cleavage (34 vs 70 and 78%) and blastocyst (3 vs 27 and 31%) rates were significantly lower for the buffalo than for the transported and non-transported domestic cattle, respectively.Fil: Konrad, José Luis. Universidad Nacional del Nordeste. Facultad de Cs.veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Scian, R.. Pont. Universidad Catolica Arg."sta.maria de Los Bs.as.". Facultad de Cs.agrarias; ArgentinaFil: Garrido, María José. Universidad Nacional del Nordeste. Facultad de Cs.veterinarias; ArgentinaFil: Taminelli, G.. Pont. Universidad Catolica Arg."sta.maria de Los Bs.as.". Facultad de Cs.agrarias; ArgentinaFil: Sansinena, Marina Julia. Pont. Universidad Catolica Arg."sta.maria de Los Bs.as.". Facultad de Cs.agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Determinación de coeficientes de transferencia de calor en sistemas biológicos durante el enfriamiento en nitrógeno líquido considerando regímenes de ebullición en film y nucleada

El proceso de criopreservación consiste en reducir la temperatura de la muestra hasta alcanzar el punto de estabilidad biológica. En particular la medición experimental de la curva temperatura versus tiempo es fundamentalpara el cálculo de velocidades de enfriamiento y para detectarsi la muestra ha sido vitrificada o si ha sufrido un procesode cambio de fase congelación en la cual se forma cristales de hielo. Cuando un objeto se sumerge en Nitrógeno Líquido (LN2) entra en un régimen de ebullición en film debido a las grandes diferencias de temperatura entre el objeto y el LN2 T). Este fenómeno ocurre debido a que el flujo de calor desde el objeto hacia el LN2 provoca la evaporación del nitrógeno en la cercanía del dispositivo generándose una capa de vapor aislante que retarda la transferencia de energía. Este fenómeno se lo conoce como efecto “Leidenfrost”. Las urvas de flujo calórico versus T (curvas de ebullición) para dispositivos empleados en criobiologíano son frecuentes enlaliteratura; ademásdad las pequeñas dimensiones de los objetos existen limitaciones para la medición experimental de la temperatura versus tiempo. El conocimiento de esta información permite determinar los coeficientes de transferencia de calor que gobiernan el proceso de enfriamiento: ebullición en film, transición y nucleada. Para predecir los coeficientesde transferencia de calor es necesario modelar matemáticamente las ecuaciones diferenciales a derivadas parciales delatransferencia de energía aplicando condiciones de borde convectivas. En el presente trabajo se estimaronlos coeficientes de transferencia de calor y las curvas de ebullición durante el enfriamiento de pajuelas plásticas conteniendo hielo (a temperaturas iniciales entre 2ºC a 9ºC) cuando se sumergían las muestras en LN2. Estos experimentos lograron detectar dos etapas en el proceso de ebullición, produciéndoseprimero ebullición en film y luego una transición a ebullición nucleada. Se utilizó un programa en elementos finitos mediante el software COMSOL para la predicción de la curva de temperatura versus tiempoque permitió estimar los coeficientes de transferencia de calor en cada régimen de ebullición. Se realizaron experimentos independientes utilizando pajuelas plásticas que contenían un fluido biológico (semen+extender) con el fin de corroborar los valores de coeficientes de transferencia encontrados previamente. En este caso el programa tiene en cuenta las propiedades variables con la temperatura en el fluido biológico, lo cual constituye un problema matemático altamente no lineal y además un coeficiente de transferencia variable según el régimen de ebullición que ocurre en cada etapa del enfriamiento. Se realizó la validación del programa contrastando las experiencias de laboratorio con las predicciones numéricas, y se logr una excelente concordancia. La simulación computacional es una herramienta importante para la determinaciónde los coeficientes de transferencia de calor y para la optimización del proceso de criopreservacin de dispositivos que contienen fluidos de interés biológico.Publicado en Terceras Jornadas de Investigación, Transferencia y Extensión. La Plata : Universidad Nacional de La Plata, 2015.Facultad de Ingenierí