Table of contents  Problem description  Objective  Materials and methods  Results  Conclusion  Reference .

Problem description The majority of pregnancies can be achieved by Slow-freezing method   The viability of oocytes has been highly variable (ranging from 2.2 to 64. vitrified bovine oocytes in conventional straws achieved results better than those observed VITRIFICATION with the slow freezing method OPSs : can obtain more rapid cooling and warming rate .0%) Ice formation damaged the cells Otoi et al.

.Objective To explored the effects of vitrification of using conventional straws and OPS on morphological survival. and chromosomes. the meiotic spindles.

MATERIALS AND METHODS 10 IU of Gonadotrophi Preparation of oocytes trigger ovulation After 50hrs (inject the mice again with 10 IU of Gonadotrophin) 18 hrs later Collect the mature oocytes Remove obviduct Collect cumulus oocyte complexes Remove granulosa cells of the oocytes .

 Vitrification using conventional straws  Vitrification using OPS  Controls without treatment .MATERIALS AND METHODS The oocytes were allocated to four groups:  Exposure to cryoprotectants without cooling.

125 mol/l sucrose in a 4-well dish. 0. The vitrification solution: 5.5 mol/l ethylene glycol for 5 min transferred into a drop on a Petri and mixed for equilibration the oocytes were transferred into 0.5.125 mol/l sucrose.5. and incubated at 37°C.5 mol/l ethylene glycol and 1.25 and 0. 0.MATERIALS AND METHODS  Preparation of pretreatment. transferred into the culture medium. for 2.25 and 0.  . vitrification and dilution solutions    The pretreatment solution: 1.  Oocytes exposed to cryoprotectants without cooling: Pretreate oocytes with 1.5 min in each solution at 37°C  washed.5mol/l ethylene glycol.0mol/l sucrose The solutions for dilution: 0.

2 cm of vitrification medium containing oocytes.MATERIALS AND METHODS  Vitrification of oocytes in conventional straws     Pretreated oocytes with 1.5.5 mol/l ethylene glycol and exposed to EG5.25 ml plastic straw. 0. 0. and 3.5 Load into a 0. The straw was filled with 1 cm of vitrification medium.5 cm of air. .5 cm of vitrification medium The straw was plunged into liquid nitrogen at 1 min after the oocytes had had contact with EG5.5 cm of air.

the OPS were held in the air for 5 s.5 mol/l sucrose. They were then diluted and incubated as above. .5 . The vitrification medium became liquefied.5 mol/l ethylene glycol and then mixed with EG5. and the tip was put into a drop (400 µl) of 0. and the oocytes were expelled from the OPS. Loading into the tip of the OPS Plunging the OPS into liquid nitrogen At warming.MATERIALS AND METHODS simply touching a microdrop of vitrification solution containing oocytes  Vitrification of oocytes in OPS      The oocytes were pretreated with 1.

 Washed in 0.01% Tween-20 (Merck) for 15 min.  The oocytes were then incubated in anti.5% bovine serum albumin (BSA) for 45 min. Fluorescent staining of meiotic spindles and chromosomes  Preservation of oocytes was achieved by fixation in 2% formaldehyde with 0.  Observation of spindles and chromosomes  Fluorescence was observed using a microscope with a magnification of x400.  The oocytes were transferred into DPBS with 0. Excess antibody and dye were washed out in 0.-tubulin monoclonal antibody (Sigma) in DPBS with 0.01% Tween-20 for 15 min.02% Triton X-100 in DPBS at 37°C for 30 min. .  Tubulin staining intensity was amplified by incubating the oocytes in fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (Sigma) for 45 min together with Hoechst 33258 (20 µg/ml) (Sigma) which stained the chromatin.5% BSA for 60 min and then wet mounted.

disruption of the spindle. Dispersion of chromosomes was defined as abnormal .Assessment of spindle morphology and chromosome arrangement    Normal spindle morphology was barrel-shaped with microtubules traversing between both poles and chromosomes Abnormal spindle morphology included a reduction of the number of microtubules or the size of the spindle. or complete absence of a spindle.

RESULTS .

of oocytes Intact morphology  The survival rate decreased Exposed 118 118(100%) Straws 161 130 (81%) after vitrification either in conventional straws or in OPS -->more oocytes were damaged in the OPS group than in the conventional straw group. OPS 177 109(62%) Need to improve .Morphological survival of mouse oocytes Treatments No.

of oocyte s 59 Normal spindles 56(95%) Reduced spindles 3 (5) Disrupte d spindles 0(0) 1(2) Absent spindles 0(0) 31(56)  The majority of the Control Exposed 55 0(0) 23(42) Straw 63 0(0) 17(27) 6(10) 40(63) OPS 54 0(0) 19(35) 3(6) 32(59) control oocytes (95%) contained a normal spindle  Non having normal spindles for Oocytes exposed to cryoprotectants or vitrified either in conventional straws or in OPS .Spindle morphology Conditions No.

of oocytes 57 Normal spindles 55(96) Reduced spindles 2 (4) Disrupte d spindles 0(0) Absent spindles 0(0)  spindles morphology of Exposed 63 53(84) 10(16) 0(0) 0(0) Straw 67 14(21) 45(67) 3(4) 5(8) OPS 55 43(78) 10(18) 2(4) 0(0) control not change  Oocytes exposed or vitrified either in conventional straws or in OPS had significantly smaller percentages of normal spindles than the controls.Spindle morphology after 1h incubation Conditio ns Control No.  A significantly smaller percentage of normal spindles were restored in oocytes vitrified in conventional straws than in oocytes exposed and those vitrified in OPS  Reduced spindles abnormal patterns .

of oocytes Compact Disperse chromos d omes chromos omes 58(98) 1(2) After 1h incubation Conditio ns No. of oocytes Compact Disperse chromos d omes chromos omes Control 59 Control 57 56(98) 1(2) Exposed 55 45(81) 10(19) 47(75) 16(25) 42(78) 12(22) Exposed 63 60(95) 3(5) 52(78) 15(22) 48(87) 7(13) Straw 63 Straw 67 OPS 54 OPS 55 .Chromosomal patterns Conditio ns No.

 Oocytes vitrified in OPS had more normal spindles restored than did those in conventional straws.Conclusion  Mouse oocytes vitrified in OPS had a smaller percentage of morphological survival than those in conventional straws.  OPS tended to have a greater percentages of compact chromosomes than the conventional straw group. .

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