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

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.

Convective heat transfer coefficients of open and closed Cryotop® systems under different warming conditions.

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/m2K. 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.

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 (<3mm) and large (>8mm) diameter follicles available per OPU, a higher proportion of medium-sized follicles (3-8mm) 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.

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

BACKGROUND: Dry ice-ethanol bath (-78 degree C) 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.

DETERMINATION OF HEAT TRANSFER COEFFICIENTS FOR FRENCH PLASTIC SEMEN STRAW SUSPENDED IN STATIC NITROGEN VAPOR OVER LIQUID NITROGEN.

BACKGROUND: The use of mathematical models describing heat transfer during the freezing process is useful for the improvement of cryopreservation protocols. A widespread practice for cryopreservation of spermatozoa of domestic animal species consists of suspending plastic straws in nitrogen vapor before plunging into liquid nitrogen. Knowledge of surface heat transfer coefficient (h) is mandatory for computational modelling; however, h values for nitrogen vapor are not available. OBJECTIVE: In the present study, surface heat transfer coefficients for plastic French straws immersed in nitrogen vapor over liquid nitrogen was determined; vertical and horizontal positions were considered. MATERIALS AND METHODS: Heat transfer coefficients were determined from the measurement of time-temperature curves and from numerical solution of heat transfer partial differential equation under transient conditions using finite elements. The h values experimentally obtained for horizontal and vertically placed straws were compared to those calculated using correlations based on the Nusselt number for natural convection. RESULTS: For horizontal straws the average obtained value was h=12.5 ± 1.2 W m(2) K and in the case of vertical straws h=16 ± 2.48 W m(2) K. The numerical simulation validated against experimental measurements, combined with accurate h values provides a reliable tool for the prediction of freezing curves of semen-filled straws immersed in nitrogen vapor. CONCLUSION: The present study contributes to the understanding of the cryopreservation techniques for sperm freezing based on engineering concepts, improving the cooling protocols and the manipulation of the straws.

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

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/m(2) K and for nucleate-boiling h=1355±51 W/m(2) 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 field.

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 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.

How important are internal temperature gradients in french straws during freezing of bovine sperm in nitrogen vapor?

The subject of present work was to predict internal temperature gradients developed during freezing of bovine sperm diluted in extender, packaged in 0.5 ml French plastic straws and suspended in static liquid nitrogen vapor at -100 degree C. For this purpose, a mathematical heat transfer model previously developed to predict freezing times (phase change was considered) of semen/extender packaged in straw was extended to predict internal temperature gradients during the cooling/freezing process. Results showed maximum temperature differences between the centre and the periphery of semen/extender "liquid" column was 1.5 degree C for an external heat transfer coefficient, h = 15 W per (m(2) K), and only 0.5 degree C for h = 5 W per (m(2) K). It is concluded that if a thermocouple wire were inserted in a 0.5 ml plastic straw to monitor the freezing process in nitrogen vapor, its radial position would have little importance since expected internal gradients may be safely neglected. This finding facilitates the interpretation of freezing rates in 0.5 ml plastic straws immersed in nitrogen vapor over liquid nitrogen, a widely used method for cryopreservation of bovine spermatozoa.

Mathematical prediction of freezing times of bovine semen in straws placed in static vapor over liquid nitrogen.

A widespread practice in cryopreservation is to freeze spermatozoa by suspending the straws in stagnant nitrogen vapor over liquid nitrogen (N(2)V/LN(2)) for variable periods of time before plunging into liquid nitrogen (-196°C) for indefinite storage. A mathematical heat transfer model was developed to predict freezing times (phase change was considered) required for bull semen and extender packaged in 0.5ml plastic straws and suspended in static liquid nitrogen vapor. Thermophysical properties (i.e. thermal conductivity, specific heat, density, initial freezing temperature) of bovine semen and extender as a function of temperature were determined considering the water change of phase. The non-stationary heat transfer partial differential equations with variable properties (nonlinear mathematical problem) were numerically solved considering in series thermal resistances (semen suspension-straw) and the temperature profiles were obtained for both semen suspension and plastic straw. It was observed both the external heat transfer coefficient in stagnant nitrogen vapor and its temperature (controlled by the distance from the surface of liquid nitrogen to the straw) affected freezing times. The accuracy of the model to estimate freezing times of the straws was further confirmed by comparing with experimental literature data. Results of this study will be useful to select "safe" holding times of bull semen in plastic straws placed N(2)V/LN(2) to ensure that complete freezing of the sample has occurred in the nitrogen vapor and avoid cryodamage when plunging in LN(2). Freezing times predicted by the numerical model can be applied to optimize freezing protocols of bull semen in straws.

Assessment of external heat transfer coefficient during oocyte vitrification in liquid and slush nitrogen using numerical simulations to determine cooling rates.

In oocyte vitrification, plunging directly into liquid nitrogen favor film boiling and strong nitrogen vaporization. A survey of literature values of heat transfer coefficients (h) for film boiling of small metal objects with different geometries plunged in liquid nitrogen revealed values between 125 to 1000 W per per square m per K. These h values were used in a numerical simulation of cooling rates of two oocyte vitrification devices (open-pulled straw and Cryotop), plunged in liquid and slush nitrogen conditions. Heat conduction equation with convective boundary condition was considered a linear mathematical problem and was solved using the finite element method applying the variational formulation. COMSOL Multiphysics was used to simulate the cooling process of the systems. Predicted cooling rates for OPS and Cryotop when cooled at -196 degree C (liquid nitrogen) or -207 degree C (average for slush nitrogen) for heat transfer coefficients estimated to be representative of film boiling, indicated lowering the cooling temperature produces only a maximum 10 percent increase in cooling rates; confirming the main benefit of plunging in slush over liquid nitrogen does not arise from their temperature difference. Numerical simulations also demonstrated that a hypothetical four-fold increase in the cooling rate of vitrification devices when plunging in slush nitrogen would be explained by an increase in heat transfer coefficient. This improvement in heat transfer (i.e., high cooling rates) in slush nitrogen is attributed to less or null film boiling when a sample is placed in slush (mixture of liquid and solid nitrogen) because it first melts the solid nitrogen before causing the liquid to boil and form a film.

Comparison of heat transfer in liquid and slush nitrogen by numerical simulation of cooling rates for French straws used for sperm cryopreservation.

Slush nitrogen (SN(2)) is a mixture of solid nitrogen and liquid nitrogen, with an average temperature of -207 °C. To investigate whether plunging a French plastic straw (commonly used for sperm cryopreservation) in SN(2) substantially increases cooling rates with respect to liquid nitrogen (LN(2)), a numerical simulation of the heat conduction equation with convective boundary condition was used to predict cooling rates. Calculations performed using heat transfer coefficients in the range of film boiling confirmed the main benefit of plunging a straw in slush over LN(2) did not arise from their temperature difference (-207 vs. -196 °C), but rather from an increase in the external heat transfer coefficient. Numerical simulations using high heat transfer (h) coefficients (assumed to prevail in SN(2)) suggested that plunging in SN(2) would increase cooling rates of French straw. This increase of cooling rates was attributed to a less or null film boiling responsible for low heat transfer coefficients in liquid nitrogen when the straw is placed in the solid-liquid mixture or slush. In addition, predicted cooling rates of French straws in SN(2) tended to level-off for high h values, suggesting heat transfer was dictated by heat conduction within the liquid filled plastic straw.

Numerical simulation of cooling rates in vitrification systems used for oocyte cryopreservation.

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.

In vitro production of llama (Lama glama) embryos by intracytoplasmic sperm injection: effect of chemical activation treatments and culture conditions.

Assisted reproductive technologies in the llama (Lama glama) are needed to provide alternative methods for the propagation, selection and genetic improvement; however, recovery of adequate quantity and quality of spermatozoa for conventional IVF is problematic. Therefore, an effort was made to adapt the intracytoplasmic sperm injection (ICSI) procedure for the in vitro production of llama embryos. The specific objectives of this study were: (1) to determine in vitro maturation rates of oocytes recovered by transvaginal ultrasound-guided oocyte aspiration (TUGA) or flank laparotomy; (2) to evaluate the effects of activation treatments following ICSI; (3) to evaluate the development of llama ICSI embryos in CR1aa medium or in an oviduct cell co-culture system. Llamas were superstimulated by double dominant follicle reduction followed by oFSH administered in daily descending doses over a 3-day interval. Oocytes were harvested by flank laparotomy or TUGA and matured in vitro for 30 h. Mature oocytes were subjected to ICSI followed by no chemical activation (Treatment A), ionomycin only (Treatment B) or ionomycin/DMAP activation (Treatment C). More oocytes were recovered by flank laparotomy procedure compared with TUGA (94% versus 61%, P<0.05) and a greater number of oocytes harvested by flank laparotomy reached the metaphase-II stage (77% versus 44%, P<0.05). After ICSI, the proportion of cleaved and 4-8-cell stages embryos was significantly greater when injected oocytes were activated with ionomycin/DMAP combination (63% and 38%, respectively, P<0.05). The co-culture of ICSI embryos with llama oviduct epithelial cells resulted in progression to morula (25%) and blastocyst (12%) stages; whereas, all embryos cultured in CR1aa medium arrested at the 8-16-cell developmental stage.

Banteng (Bos javanicus) embryos and pregnancies produced by interspecies nuclear transfer.

The banteng (Bos javanicus), a member of the bovidae family, is currently listed as threatened by the IUCN Red List and it is estimated the total world population is <10,000 animals. In exotic or endangered species, the lack of oocytes and recipients precludes the use of traditional somatic cell nuclear transfer (NT), and an approach such as interspecies NT may be the only alternative to produce embryos and offspring. A total of 348 enucleated domestic bovine oocytes were reconstructed with either male (Treatment A) or female (Treatment B) adult banteng fibroblasts and a total of 103 bovine oocytes were parthenogenically activated as a control (Treatment C). There was no significant difference in fusion rate (68 versus 77%) between Treatments A and B. Of fused couplets, those in Treatment A had greater (P < 0.05) cleavage (67 versus 51%) and blastocyst (28 versus 15%) rate than Treatment B. Of a total of 24 blastocysts transferred into 12 domestic cattle recipients from Treatment A, two pregnancies (17%) were established with heart beats detectable at 30 day by rectal ultrasonography. No pregnancies resulted from the transfer of 14 blastocysts from Treatment B. Both pregnancies were subsequently lost, one between 30 and 60 days and the second between 60 and 90 days of gestation. The bovine cytoplast supported mitotic cleavage of banteng karyoplasts, and was capable of reprogramming the nucleus to achieve blastocyst stage embryos and pregnancies in exotic bovids.