Oocyte cryopreservation is of key importance in the preservation and propagation of germplasm. Interest in oocyte cryopreservation has increased in recent years due to the application of assisted reproductive technologies in farm animals such as in vitro fertilization, nuclear transfer and the need for the establishment of ova/gene banks worldwide. However, the cryopreservation of the female gamete has been met with limited success mainly due to its small surface-area:volume ratio. In the past decade, several vitrification devices such as open pulled straws (OPS), fine and ultra fine pipette tips, nylon loops and polyethylene films have been introduced in order to manipulate minimal volumes and achieve high cooling rates. However, experimental comparison of cooling rates presents difficulties mainly because of the reduced size of these systems. To circumvent this limitation, a numerical simulation of cooling rates of various vitrification systems immersed in liquid nitrogen was conducted solving the non-stationary heat transfer partial differential equation using finite element method. Results indicate the nylon loop (Cryoloop®) is the most efficient heat transfer system analyzed, with a predicted cooling rate of 180,000°C/min for an external heat transfer coefficient h= 1000 W/m(2)K when cooling from 20 to -130°C; in contrast, the open pulled straw method (OPS) showed the lowest performance with a cooling rate of 5521°C/min considering the same value of external heat transfer coefficient. Predicted cooling rates of Miniflex® and Cryotop® (polyethylene film system) were 6164 and 37,500°C/min, respectively, for the same heat transfer coefficient.

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Although interspecies somatic cell nuclear transfer (iSCNT) has potential applications in the conservation of exotic species, an in vitro developmental block has been observed in embryos produced by this approach. It has been suggested that mitochondrial mismatch between donor cell and recipient oocyte could cause embryonic developmental arrest. A series of experiments was conducted to investigate the effect of mixed mitochondrial populations (heteroplasmy) on early development of iSCNT-derived cloned embryos. The effect of combining the techniques of ooplasm transfer (OT) and somatic cell nuclear transfer (SCNT) was examined by monitoring in vitro embryonic development; the presence and pattern of migration of foreign mitochondria after OT was analysed by MitoTracker staining. In addition, the effect of transferring caprine ooplasm (iOT) into the bovine enucleated oocytes used in iSCNT was analysed. There was no significant effect of the sequence of events (OT-SCNT or SCNT-OT) on the number of fused, cleaved, blastocyst or hatched blastocyst stage embryos. MitoTracker Green staining of donor oocytes used for OT confirmed the introduction of foreign mitochondria. The distribution pattern of transferred mitochondria most commonly remained in a distinct cluster after 12, 74 and 144 h of in vitro culture. When goat ooplasm was injected into bovine enucleated oocytes (iSCNT), there was a reduction (p < 0.05) in fusion (52 vs. 82%) and subsequent cleavage rates (55 vs. 78%). The procedure of iOT prior to iSCNT had no effect in overcoming the 8- to 16-cell in vitro developmental block, and only parthenogenetic cow and goat controls reached the blastocyst (36 and 32%) and hatched blastocyst (25 and 12%) stages, respectively. This study indicates that when foreign mitochondria are introduced at the time of OT, these organelles tend to remain as distinct clusters without relocation after a few mitotic divisions. Although the bovine cytoplast appears capable of supporting mitotic divisions after iOT-iSCNT, heteroplasmy or mitochondrial incompatibilities may affect nuclear-ooplasmic events occurring at the time of genomic activation.

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The warming of cryopreserved samples supported by small volume devices is governed by heat transfer phenomena which are mathematically described by the solution of the transient heat conduction partial differential equations; the convective heat transfer coefficient (h) is an important parameter involved in the boundary condition which is related to the fluid dynamic behavior at the interface device-warming fluid (water, sucrose solution or air). Unfortunately, h values for small volume devices (i.e. Cryotop) have not been experimentally determined. Moreover, heat transfer coefficients during warming of Cryotop cannot be obtained through classical dimensionless correlations expressed in terms of Nusselt vs. Reynolds and Prandtl numbers that are available for regular geometries and single materials. It is the purpose of present work to determine the convective heat transfer coefficients (h) by numerically solving the heat transfer equation applying the finite element method. Numerical simulations allowed to predict time-temperature histories and warming rates under different protocols in Cryotop system which were compared with literature warming rates reported for this device. The h values were calculated considering the heterogeneous structure of the domain (microdrop, plastic-support) and the irregular three-dimensional geometry. The warming conditions analyzed were: a) open system in contact with air and sucrose solution at 23 °C) and b) closed system in contact with air and water at 23 °C. The h values of the Cryotop open system immersed in sucrose solution (23 °C), that according to literature achieved a survival in the order of 80%, are in the range of 1800-2200 W/mK. The h values obtained in this work for warming conditions are critical parameters for cryobiologists when studying heat transfer rate in this small volume device.

To date, there have been no reports of somatic cell nuclear transfer in llamas. The application of this methodology to the camelid industry could be helpful in the propagation of genetically valuable animals. The objective of this study was to produce nuclear transfer llama embryos comparing the development of these llama embryos cultured in either CR1aa medium (treatment A) or G1.2 medium (treatment B) medium. Llamas were superstimulated by double dominant follicle reduction 12 days apart, followed by pFSH administered in daily descending doses over a 3-day interval (total dose of 200 mg). Animals were ovariectomized by flank laparotomy, follicles were aspirated from excised ovaries and oocytes were in vitro matured for a 30-h period. Adult female llama fibroblasts were used as donor karyoplasts and injected into enucleated llama oocytes. Embryo development was assessed after 2 days of culture. A total of 307 follicles were aspirated from nine treated females, resulting in 298 (97%) oocytes recovered. Of a total of 229 evaluated oocytes, 120 (52%) achieved nuclear maturation. Of a total of 80 reconstructed couplets, 50 (62.5%) were successfully fused. Subsequent cleavage rates were 32 and 40% for treatments A and B, respectively, with no significant difference (p < 0.05) detected between treatment groups. A total of 11 embryos (8-cell to morula stages) were transferred to synchronized recipient llamas. Ultrasonography at 14 days post-transfer indicated that no pregnancies were established. This study shows that nuclear transfer can be successfully applied to the production of llama embryos. Further research is needed to identify optimal parameters to improve efficiency of nuclear transfer in this species.

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 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. 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.