Effect of Disaccharide Inclusion in Vitrification and Warming Solutions on Developmental Competence of Vitrified/Warmed Germinal Vesicle Stage Buffalo Oocytes.

BACKGROUND: Cryopreservation of immature oocyte is a potential strategy for preserving the female germline, providing a non-seasonal, easily accessible source for reproduction and science. Exposure of oocytes to high concentrations of cryoprotectants during vitrification is toxic and can negatively impact the fertilization ability and development of vitrified/warmed oocytes. OBJECTIVE: 1) to evaluate the effects of exposure of buffalo germinal vesicle (GV) oocytes to different vitrification solutions (VS), either supplemented with or without sucrose, on cumulus expansion and nuclear maturation following IVM; and 2) to compare the effects of sucrose and trehalose in the warming solution on developmental competence of buffalo oocytes vitrified at the GV-stage. MATERIALS AND METHODS: Cumulus oocyte complexes (COCs) obtained at slaughter from mature buffalo ovaries were randomly assigned into five groups: control - directly subjected to IVM); VS1 group - exposed to 20% ethylene glycol (EG) + 20% glycerol (GLY) + 0.5 M sucrose; VS2 group - exposed to 20% EG + 20% GLY; VS3 group - subjected to 20% EG+20% dimethyl sulfoxide (DMSO) + 0.5 M sucrose; and VS4 group - subjected to 20% EG+20% DMSO. Following cryoprotectant dilution, viable oocytes were matured in vitro for 22 h; cumulus expansion and nuclear maturation were then evaluated (Experiment 1). COCs were vitrified by solid surface vitrification (SSV) in a solution composed of 20% EG + 20% DMSO (VS4). Following vitrification, COCs were warmed in a solution composed of either sucrose or trehalose in decreasing concentrations (1 M, 0.5 M and 0.25 M). Morphologically viable oocytes were matured, fertilized and cultured in vitro. Cleavage and blastocyst rates were evaluated at 30 h and day 7 post-insemination (p.i.), respectively (Experiment 2). RESULTS: Exposure of GV-buffalo oocytes to different cryoprotectant combinations did not significantly affect cumulus expansion following IVM. However, nuclear maturation rate (oocytes at MII) was significantly higher (P<0.05) in the groups exposed to sucrose-free vitrification solutions (VS2 and VS4) and not significantly different from the control. Compared with the control group, the cleavage and blastocyst rates were significantly (P<0.05) lower in oocytes vitrified and then warmed in a solution containing trehalose; whilst this was not the case when sucrose was present in the solution. CONCLUSION: Our results suggest that exposure of buffalo GV-oocytes to sucrose-free vitrification solutions improved nuclear maturation after IVM. Moreover, warming of vitrified buffalo oocytes in sucrose-based solution improved preimplantation development following IVM and IVF compared to trehalose based media.

Effect of oviduct and follicular fluids on ram sperm capacitation and acrosome reaction in vitro.

The present study was designed to study the influence of different concentrations of oviduct fluid (OF) and follicular fluid (FF) on ram sperm capacitation and acrosome reaction in vitro. Forty semen samples were collected from three Barki rams throughout the period of study (10 weeks). Fresh semen was evaluated, layered under S-TALP (sperm Tyrode's albumin lactate pyruvate) medium and subjected to swim up techniques. Split fractions of semen were incubated in media enriched with different levels of OF (10, 20, 40, 50 and 75 µL/mL) or FF (10, 20, 40, 50, 100, 150 and 200 µL/mL). Best concentrations were compared and used to evaluate the ram sperm functions including progressive motility, hyperactivity and acrosome reaction. The present findings showed a significant increase in individual motility percentage (IM %) when ram spermatozoa were treated with 10 and 50 µL of oviduct fluids as compared to the other treatments after 1 h of incubation. Addition of 50 µL/mL of OF or FF had beneficial effect on sperm hyperactivity after 2 and 1 h incubation respectively. Furthermore, addition of 50 µL/mL oviduct fluid to ram sperm maintained significantly (P < .05) higher total acrosome reaction (AR %) after 3 h of incubations than those observed in other groups. In conclusion, treatment of ram spermatozoa with 50 µL/mL of OF or 40 µL/mL of FF for 3-4 h incubation respectively was considered the best level of oviduct or follicular fluid to be used for IVF.