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.

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.

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.

APC senses cell-cell contacts and moves to the nucleus upon their disruption.

The adenomatous polyposis coli (APC) tumor suppressor protein is involved in the Wnt/wingless pathway, modulating beta-catenin activity. We report the development of a highly specific, chemically synthesized oligobody (oligonucleotide-based synthetic antibody), directed against the N-terminal region of APC. Using this reagent, we found that within 16 h of disrupting HT-29 cell-cell contacts by harvesting cells with trypsin/EDTA treatment and replating, APC was translocated from the cytoplasm to the nucleus. Five days after plating the cells, when the cells had returned to their normal confluent phenotype and cell-cell contacts were reestablished, APC returned to the cytoplasm. These results suggest that APC functions as part of a "sensor" system, and responds to the loss of cell-cell contacts by moving to the nucleus, and returning to the cytoplasm when the contacts are fully restored.