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

Implications of glass transition in the devitrification process and storage management of vitrified oocytes and embryos

Abstract: 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 vitrification carrier (open-pulled straw, OPS) was conducted and the implications on specimen storage in nitrogen vapor phase were determined. The time required for initiation of devitrification was determined by mathematical modeling and compared with temperatures in the vapor phase of liquid nitrogen cryogenic dewars. Results indicated that the glass transition ranged from -126 to -121ºC and devitrification was initiated at -109ºC. Interestingly, samples entered rubbery state at -121ºC and therefore could potentially initiate devitrification above this value, with the consequent damaging effects to cell survival. Devitrification itimes were mathematically modeled considering an initial temperature of material immersed in liquid nitrogen (-196ºC) and two arbitrarily selected temperatures (-50 and -70ºC) to which a sample could be exposed at the neck of dewar. The mathematical model indicated samples could reach glass transition temperatures and undergo devitrification in 30 seconds. 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. Due to the reduced time handling period before samples reach critical rubbery and devitrification values, caution should be exercised when handling samples in vapor phas

Producción de embriones de búfalo por fertilización in vitro luego de la maduración de los ovocitos durante el transporte prolongado

Resumen: 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 dificultades 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 transporteAbstract: The water buffalo (Bubalus bubalis) is a species with excellent adaptation to flood-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 final 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, respectivel

Establishment of pregnancies by collapse and vitrification of expanded equine blastocysts (>900 um) using a novel, microneedle and microwell-assisted puncture

Resumen: La criopreservación del embrión equino es una herramienta importante para el diferimiento de los transplantes embrionarios y la comercialización de genética. Los reportes de preñeces logradas con embriones de diámetros ≥300μm son escasos1,2. Debido a que el lavaje (d 7-9 post-inseminación) resulta generalmente en blastocistos de gran volumen (500-2000 μm), es necesario un colapso total o parcial del blastocele para evitar cristalización durante el proceso de vitrificación. Este colapso, generalmente realizado mediante equipamiento complejo de micromanipulación, es de difícil implementación en condiciones de campo. El objetivo de éste ensayo preliminar fue desarrollar un sistema de reducción de volumen sencillo y efectivo, que permita el colapso del embrión equino previo a la vitrificación

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.Centro de Investigación y Desarrollo en Criotecnología de Alimento

Mitochondrial function, blastocyst development and live foals born after ICSI of immature 4 vitrified/warmed equine oocytes matured with or without melatonin

Abstract: Oocyte vitrification is considered experimental in the horse with only three live foals reported. The oxidative conditions induced by vitrification could in part explain the poor results and melatonin, a powerful antioxidant, could stimulate ROS metabolization and restore mitochondrial function in these oocytes. Our objective was to determine the oxidative status of vitrified equine oocytes and to analyze the effect of melatonin on mitochondrial-specific ROS (mROS), oocyte maturation, ICSI embryo development and viability. Immature, abattoir-derived oocytes were held for 15 h and vitrified in a final concentration of 20% EG, 20% DMSO and 0.65 M trehalose. In Experiment 1, overall ROS was determined by DCHF-DA; vitrification increased ROS production compared to non-vitrified controls (1.29 ± 0.22 vs 0.74 ± 0.25 a. u.; P = 0.0156). In Experiment 2, mROS was analyzed by MitoSOX™ in vitrified/warmed oocytes matured with (+) or without (−) supplementation of 10−9 M melatonin; mROS decreased in vitrified and non-vitrified oocytes matured in presence of melatonin (P < 0.05). In Experiment 3, we assessed the effect of melatonin supplementation on oocyte maturation, embryo development after ICSI, and viability by pregnancy establishment. Melatonin did not improve oocyte maturation, cleavage or blastocyst rate of non-vitrified oocytes. However, vitrified melatonin (+) oocytes reached similar cleavage (61, 75 and 77%, respectively) and blastocyst rate (15, 29 and 26%, respectively) than non-vitrified, melatonin (+) and (−) oocytes. Vitrified, melatonin (−) oocytes had lower cleavage (46%) and blastocyst rate (9%) compared to non-vitrified groups (P < 0.05), but no significant differences were observed when compared to vitrified melatonin (+). Although the lack of available recipients precluded the transfer of every blastocyst produced in our study, transferred embryos from non-vitrified oocytes resulted in 50 and 83% pregnancy rates while embryos from vitrified oocytes resulted in 17 and 33% pregnancy rates, from melatonin (+) and (−) treatments respectively. Two healthy foals, one colt from melatonin (+) and one filly from melatonin (−) treatment, were born from vitrified/warmed oocytes. Gestation lengths (considering day 0 = day of ICSI) were 338 days for the colt and 329 days for the filly, respectively. Our work showed for the first time that in the horse, as in other species, intracellular reactive oxygen species are increased by the process of vitrification. Melatonin was useful in reducing mitochondrial-related ROS and improving ICSI embryo development, although the lower pregnancy rate in presence of melatonin should be further analyzed in future studies. To our knowledge this is the first report of melatonin supplementation to an in vitro embryo culture system and its use to improve embryo developmental competence of vitrified oocytes following ICSI

Ovum pick-up interval in buffalo (Bubalus bubalis) managed under wetland conditions in Argentina: Effect on follicular population, oocyte recovery, and in vitro embryo development

The excellent adaptation of water buffalo (Bubalis bubalis) to swampy environments means that animals are frequently managed in areas with restricted access for reproductive procedures. The objective of this study was to evaluate the effect of the ovum pick-up (OPU) interval on follicular population, oocyte recovery, oocyte quality and in vitro embryo production. Twelve Murrah buffaloes were subjected to two consecutive dominant follicle reductions, and randomly assigned to either 7-day (n = 6) or 14-day (n = 6) OPU interval groups. Although there was no significant difference in the average number of small (8 mm) diameter follicles available per OPU, a higher proportion of medium-sized follicles (3–8 mm) were observed in the 14-day interval group (5.129 vs 3.267; p < 0.05). The number of recovered oocytes per donor was also significantly higher (4.51 vs. 2.8; p < 0.05) in the 14-day interval group, although this was attributed to an increase in the proportion of lower quality oocytes (grades III and IV). After in vitro fertilization, embryo developmental competence from grade I and II oocytes was superior to that from grade III and IV oocytes, irrespective of OPU interval group. There was no significant difference in the proportion of grade I and II oocytes cleaved after sperm co-incubation; however, there was a higher proportion of blastocysts produced in 14-day interval group (28 vs. 6%, p < 0.05). No blastocysts were produced from grade III and IV oocytes. This study indicates it is possible to use a 14-day interval for oocyte collection in water buffalo; this approach could be considered as an alternative when access to animals is restricted.Fil: Konrad, José Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; ArgentinaFil: Clérico, Gabriel José. Pontificia Universidad Católica Argentina ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garrido, María José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; ArgentinaFil: Taminelli, Guillermo Luis. Pontificia Universidad Católica Argentina ; ArgentinaFil: Yuponi, M.. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; ArgentinaFil: Yuponi, R.. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; ArgentinaFil: Crudeli, Gustavo Angel. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; ArgentinaFil: Sansinena, Marina Julia. Pontificia Universidad Católica Argentina ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Effect of oocyte in vitro maturation interval on subsequent ICSI embryo quality and development

Introduction: Demand for in vitro equine embryo production by ICSI has increased in clinical reproduction over the last 5 years. However, the efficiency of this technique remains variable with reported blastocyst rates between 10 and 41%. Oocytes recovered by transvaginal follicular aspiration from donor mares are valuable and limited, and they are often collected from immature follicles. These oocytes require further in vitro maturation in order to be competent to be fertilized and reach the blastocyst stage. For this reason, it is crucial to determine the optimal time to perform ICSI after onset of oocyte maturation. Different in vitro maturation intervals have been reported to produce ICSI embryos in horses (28-30h[1]; 36 h-[2]; 38 h [3]; 20-22 h- [4]). However, there is no consensus about the optimal maturation interval to produce high quality embryos. An important aspect of preimplantation embryo development is cell differentiation, with the formation of the inner cell mass (ICM) and the trophectoderm (TE). The Hippo signaling pathway has been shown to play an important role in mice and cattle [5,6], but it has not been described in horses to date. Nuclear localization of a protein called Yes-associated protein 1 (YAP1) in TE cells affects the expression of CDX2 and subsequent cell differentiation. YAP1 functions as a critical co-activator for TE development in the nucleus of TE cells, but in the inner cell mass, it stays mainly in the cytoplasm as a phosphorylated inactive form [6]. In this study, we aimed to evaluate developmental rates and quality of ICSI embryos produced from oocytes which exhibited extrusion of first polar body early in IVM (Group I, 22 to 24 h) or late (Group II, 28 to 30 h). Blastocyst development and size were recorded. Embryo quality was assesed by analysis of apoptotic cells (TUNEL assay), and expression and distribution of YAP-1 by immunoflourescence (IF)