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.

In vitro development of morulae from immature caprine oocytes.

The effects of medium supplementation with oestrous goat serum and glycoprotein hormones on caprine oocyte maturation in vitro (IVM) were evidenced by proportions of resulting ova completing in vitro fertilisation (IVF) and development to the morula stage. Oocyte-cumulus complexes (OCCs) were harvested in follicular fluid from 2-5 mm diameter follicles. Oocyte maturation took place during 27 h in TCM-199 supplemented with 20% oestrous goat serum, oestradiol-17 beta (1.0 microgram/ml), and either (a) 0.5 microgram FSH/ml, (b) 100 micrograms LH/ml, (c) 100 micrograms LH + 0.5 microgram FSH/ml, (d) 100 micrograms hCG + 0.5 microgram FSH/ml, (e) 0.5 microgram TSH/ml or (f) no added glycoprotein hormone (control). Of 353 immature oocytes cultured in seven experiments, 311 (88.1%) exhibited cumulus expansion at the end of the IVM interval; all normal-appearing OCCs were inseminated. In vitro insemination was with ejaculated sperm treated with heparin (10 micrograms/ml) and caffeine (0.4 microgram/ml). Proportions (%) of inseminated ova that were fertilised (cleaved) and that reached the morula stage after IVM with (a) FSH, (b) LH, (c) LH+FSH, (d) hCG + FSH, (e) TSH and (f) no added glycoprotein hormone were (a) 22/52 (42.3%) and 9/52 (17.3%), (b) 25/54 (46.3%) and 14/54 (25.9%), (c) 52/65 (80.0%) and 26/65 (40.0%), (d) 48/78 (61.5%) and 22/78 (28.2%), (e) 14/54 (25.9%) and 4/54 (7.4%), and (f) 11/50 (22.0%) and 1/50 (2.0%), respectively. All treatments yielded better results than IVM with no added glycoprotein hormone. After IVM with added LH+FSH higher proportions of oocytes were fertilised (p < 0.05), and higher proportions reached the morula stage (p < 0.05) when compared with other treatments.

Term development of caprine embryos derived from immature oocytes in vitro.

Ovaries were surgically removed from female goats (Toggenburg, Nubian and Saanen breeds). Oocytes were collected by follicular aspiration or after ovaries were minced, then matured in mTCM-199 with 100 microg LH+0.5 microg FSH+1.0 microg estradiol 17-beta/ml for 27 h prior to in vitro fertilization (17). Although more oocytes were made available by mincing than by aspiration, higher proportions of aspirated oocytes were fertilized and developed to morulae. Proportions that fertilized and reached morulae were 82/102 (80.4%) and 50/102 (49.0%) versus 77/126 (61.1%) and 27/126 (21.4%) for oocytes obtained by aspiration and after ovarian mincing, respectively (P<0.05). Proportions of inseminated ova undergoing cleavage and continuing development to the morula stage differed significantly (P<0.05) among 5 co-culture treatment groups, with higher proportions of cleavage (23/27, 85.2%) and morulae (14/27, 51.9%) obtained by co-culture on caprine cumulus cells (cCC). Some oocytes reached the blastocyst stage (4/54, 7.4%)following oocyte collection by aspiration and culture on caprine oviduct epithelial cells (cOEC). After 4- and 8-cell stage embryos obtained by aspiration and culture on cCC were transferred pregnancy resulted. Twin male kids (developed from different embryos) were born on August 6, 1993, and have developed into normal bucks. Conditions reported here provided an adequate environment for support of oocyte maturation, fertilization and early embryonic development in vitro (IVMFC) with normal development after embryo transfer.