Otras biotecnologías y su aplicación en el búfalo

En 1914, Hans Speaman demostró que el núcleo del embrión de salamandra tenía características pluripotenciales hasta el estado de desarrollo de 16 células (publicado Speaman, 1938). En este estudio clásico, el autor tomó un cabello de su hijo y lo utilizó para lograr una constricción en el cigoto recién fecundado de una salamandra. Mediante este simple mecanismo el investigador logró aislar el núcleo en un lado del embrión y observó que ese lado (con núcleo) continuaba con sus divisiones celulares hasta detenerse en las 16 células, mientras que el otro lado (sin núcleo) detenía su desarrollo. En este momento, Speaman permitió el paso de un único núcleo a la porción de citoplasma sin núcleo y utilizó la ligadura para seccionar el embrión totalmente. Esta nueva porción de embrión, ahora nuevamente con núcleo, procedió con su desarrollo normalmente hasta llegar al estado de larva..

Theoretical prediction of the effect of heat transfer parameters on cooling rates of liquidfilled plastic straws used for cryopreservation of spermatozoa

Abstract: Heat transfer plays a key role in cryopreservation of liquid semen in plastic straws. The effect of several parameters on the cooling rate of a liquid-filled polypropylene straw when plunged into liquid nitrogen was investigated using a theoretical model. The geometry of the straw containing the liquid was assimilated as two concentric finite cylinders of different materials: the fluid and the straw; the unsteady-state heat conduction equation for concentric cylinders was numerically solved. Parameters studied include external (convection) heat transfer coefficient (h), the thermal properties of straw manufacturing material and wall thickness. It was concluded that the single most important parameter affecting the cooling rate of a liquid column contained in a straw is the external heat transfer coefficient in LN2. Consequently, in order to attain maximum cooling rates, conditions have to be designed to obtain the highest possible heat transfer coefficient when the plastic straw is plunged in liquid nitrogen

Determination of surface heat transfer coefficients during the cryopreservation of bovine germplasm using numerical simulations

Resumen: El proceso de criopreservación de germoplasma animal consiste en reducir la temperatura de una muestra hasta alcanzar la estabilidad biológica. En particular la medición experimental de la curva temperatura vs tiempo es fundamental para el cálculo de velocidades de enfriamiento y para detectar si la muestra ha sido vitrificada o si ha sufrido un proceso de cambio de fase (congelación). Dadas las pequeñas dimensiones de los dispositivos de criopreservación, existen dificultades técnicas para la correcta medición experimental de la temperatura o el flujo calórico en función del tiempo, tanto para el enfriamiento en nitrógeno vapor como en nitrógeno líquido, siendo escasa en la literatura, la información acerca de los coeficientes de transferencia de calor (h) en estos procesos. La simulación computacional constituye una herramienta valiosa para poder obtener a partir de las historias térmicas experimentales los valores de h alimentando las propiedades térmicas adecuadas. En este sentido se estudió la criopreservación de semen bovino que habitualmente se dispone en pajuelas plásticas, las cuales se suspenden en nitrógeno vapor a los efectos de deshidratar las células, previo a su inmersión en nitrógeno líquido para su conservación. Los objetivos del presente trabajo fueron determinar coeficientes (h) durante el enfriamiento de pajuelas plásticas conteniendo germoplasma animal, tanto cuando se sumergían en nitrógeno vapor como en nitrógeno líquido a partir de los perfiles de temperatura medidos experimentalmente y de las simulaciones numéricas. Se utilizó un programa en elementos finitos con propiedades termofísicas del fluido biológico dependientes de la temperatura, incluyendo el cambio de fase del agua lo cual constituye un problema matemático complejo y altamente nolineal. La determinación de los h que gobiernan el proceso constituye un aspecto muy importante para la optimización de protocolos durante la criopreservación de material biológico.Abstract: In order to maintain cell viability, biological functions must be halted by cooling it into a solid phase. The time– temperature curve of a sample allows the calculation of cooling rates and helps to determine whether it is vitrified or undergoes phase change transition (freezing). Due to the small dimensions of the devices usually there are technical difficulties associated to the experimental measurements of time-temperature curves or heat flux for samples that are suspended in nitrogen vapor and/or immersed in liquid nitrogen. There is a lack of experimental information concerning the surface heat transfer coefficients of plastic French straws containing biological samples such as bovine semen when they are cryopreserved. A widespread practice is to freeze the sample by suspending the straws in nitrogen vapor over liquid nitrogen for variable periods before plunging into liquid nitrogen for long-term storage. The objectives of the present study were to numerically solve the heat transfer equation to determine the surface heat transfer coefficients that govern the process when straws are suspended in nitrogen vapor over liquid nitrogen and when they are directly plunged in liquid nitrogen. This represents a highly non-linear mathematical problem that must be solved numerically since the thermophysical properties that are involved in the heat transfer partial differential equations undergo abrupt changes with temperature due to ice formation. These algorithms can help optimize cryopreservation protocols which are important when trying to maximize viability of cells

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

Determination of heat transfer coefficients in plastic French straws plunged in liquid nitrogen

Abstract: The knowledge of the thermodynamic process during the cooling of reproductive biological systems is important to assess and optimize the cryopreservation procedures. The time temperature curve of a sample immersed in liquid nitrogen enables the calculation of cooling rates and helps to determine whether it is vitrified or undergoes phase change transition. When dealing with cryogenic liquids, the temperature difference between the solid and the sample is high enough to cause boiling of the liquid, and the sample can undergo different regimes such as film and/or nucleate pool boiling. In the present work, the surface heat transfer coefficients (h) for plastic French straws plunged in liquid nitrogen were determined using the measurement of time-temperature curves. When straws filled with ice were used the cooling curve showed an abrupt slope change which was attributed to the transition of film into nucleate pool boiling regime. The h value that fitted each stage of the cooling process was calculated using a numerical finite element program that solves the heat transfer partial differential equation under transient conditions. In the cooling process corresponding to film boiling regime, the h that best fitted experimental results was h=148.12 ± 5.4 W/m2 K and for nucleate-boiling h=1355 ± 51 W/m2 K. These values were further validated by predicting the time-temperature curve for French straws filled with a biological fluid system (bovine semen-extender) which undergoes freezing. Good agreement was obtained between the experimental and predicted temperature profiles, further confirming the accuracy of the h values previously determined for the ice-filled straw. These coefficients were corroborated using literature correlations. The determination of the boiling regimes that govern the cooling process when plunging straws in liquid nitrogen constitutes an important issue when trying to optimize cryopreservation procedures. Furthermore, this information can lead to improvements in the design of cooling devices in the cryobiology fiel

Physicochemical changes and sensory characterization of a balsamic vinegar dressing at different °brix

Abstract: A balsamic vinegar dressing was developed by concentrating commercial balsamic vinegar (wine vinegar + grape juice) by evaporation under controlled conditions; evaporation increased the content of glucose + fructose that was naturally present in the balsamic vinegar leading to high Brix values. The water activity (aw) and viscosity of the balsamic dressing at various °Brix were measured and modeled using previously reported equations for the behavior of fructose/glucose, which showed a good fit. A quantitative descriptive analysis was performed and samples were grouped in three clusters corresponding to low (31.1–51.2), intermediate (64.0–67.5), and high (71.8–76.0) °Brix. Samples of highest °Brix were associated with sweetness, caramel flavor, visual viscosity, and reduced sourness and acetic aroma, attributes which differentiated these samples from the control and which are considered desirable for

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

Producción in vitro de embriones en búfalas en transición reproductiva en Argentina

A pesar de que las búfalas son poliéstricas, su eficiencia reproductiva muestra amplia variación durante todo el año, presentando un patrón de estacionalidad reproductiva, con reducción de la actividad sexual en los días largos. La melatonina es una hormona secretada por la glándula pineal durante la noche y representa la señal endocrina del ritmo de luz-oscuridad en el medio ambiente, y tiene un lugar central en la regulación de la temporada de la ciclicidad ovárica en los búfalos.Con el fin de aumentar la productividad del búfalo mediante alternativas que permitan la manipulación y la actividad reproductiva en períodos de transición de la estacionalidad reproductiva, se propuso evaluar la repuesta ovárica y la recuperación de ovocitos por punción folicular in vivo y su posterior producción de embriones in vitro en búfalas tratadas con implantes de liberación lenta de melatonina. Se utilizaron 16 búfalas adultas de 11,7±2,9 años, de fertilidad comprobada, con una condición corporal de 3,7±0,4 (escala 1 a 5), y un peso de 565±78 kg. El ensayo comenzó a fines de enero, momento de transición entre el anestro estacional y la temporada de reproducción. Las búfalas fueron asignadas aleatoriamente al grupo control (sin implante, n=8) o tratadas con implantes de melatonina (n=8) de liberación continua (18 mg, 1 implante cada 50 kg de peso vivo; 11,4±1,5 implantes por búfala). Los complejos ovocito-cumulus (COCs) fueron recuperados 16 días después mediante punción folicular transvaginal, madurados in vitro durante 20 horas. Luego de la maduración, los ovocitos fueron separados del cumulus y la maduración nuclear (metafase II) fue confirmada bajo lupa estereoscópica por identificación de primer corpúsculo polar y presencia de espacio perivitelino. Los ovocitos maduros fueron fertilizados con semen bubalino, y se realizó el desarrollo embrionario in vitro en incubadora gaseada con 5% de dióxido de carbono, 5% de oxígeno, temperatura de 38,7°C y humedad del 75%, durante 6 días. Las comparaciones entre los porcentajes obtenidos se analizaron por ANOVA. Los resultados obtenidos se presentan en la Tabla 1. No se observaron diferencias significativas en la cantidad de COCs recuperados por donante entre tratamientos, en la proporción de ovocitos que alcanzaron el estadio de metafase II luego de 20 horas de maduración, ni en la proporción de embriones producidos. En conclusión, el uso de implantes de melatonina en búfalas no mejoró la competencia ovocitaria para la producción in vitro de embriones bubalinos durante la época de transición reproductiva..

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

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)