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ABSTRACT: Vitrification is a rapid freezing method in which cells/tissues are frozen in a glass state without ice crystal formation. However, vitrification of bovine oocytes is challenging due to their complex structure and sensitivity to chilling. Oocytes at the germinal vesicle (GV) stage of maturation are thought to be less prone to chromosomal and microtubular damage during cryopreservation because no spindle is present and genetic material is contained within the nucleus. However, immature oocytes are thought to be more sensitive to osmotic stress and have lower cell membrane stability than mature, metaphase II (MII) stage oocytes. The present studies aimed to validate the in vitro culture system used in our laboratory and to evaluate the effect of vitrification of bovine cumulus-oocyte complexes (COC) at different meiotic stages on their in vitro maturation (IVM), cleavage and early embryo development. Analyses were conducted on each dataset with PROC GLIMMIX in SAS using binary distribution (for yes/no response variable) and considering replicate as a random factor. In Experiment 1, meiotic progression of oocytes was evaluated at different time intervals during IVM. The following COC stages were predominantly found at different IVM time intervals: GV (89%) at 0h, GV (47%) and germinal vesicle breakdown (GVBD; 44%) at 6h, metaphase I (MI; 90%) at 12h and MII (84%) at 22h (n>62 oocytes at each time group). In Experiment 2, bovine COC at 0, 6, 12 and 22h of IVM were exposed to vitrification solution (15% dimethyl sulfoxide+15% ethylene glycol+0.5M sucrose+20% CS in TCM-199), loaded onto a cryotop device and vitrified by plunging in liquid nitrogen. Following warming (1min in 0.5M sucrose+20% CS in TCM-199), COC completed 22h of IVM and the nuclear stage was evaluated with lamin A/C-4'6-diamidino-2-phenylindole staining. Upon completion of 22h of IVM, 23, 23, 35 and 89% of oocytes from 0-, 6-, 12- and 22-h groups, respectively were detected at MII (P<0.0001). In Experiment 3, cleavage and embryo development of oocytes vitrified at 0, 12 and 22h of IVM were evaluated. The cleavage rate did not differ among vitrification groups (i.e. 14% at 0h, 17% at 12h and 14% at 22h; P=0.825). Cleavage and blastocyst rates were higher (P<0.0001) in the non-vitrified (control) group than in vitrified groups (i.e. 73 vs 15% and 22 vs 0.3%, respectively). In conclusion, the maturation kinetics validated our in vitro culture system and vitrification adversely affected the ability of bovine oocytes to undergo in vitro maturation to the MII stage, in vitro fertilization and early embryo development. Vitrification of oocytes at GV, MI and MII stages of nuclear maturation did not differ in their subsequent survivability.
Reproduction Fertility and Development 12/2011; 24(1):135. · 2.11 Impact Factor
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ABSTRACT: The objective was to investigate the effects of cryodevice, vitrification solutions, and equilibration time on in vitro maturation, cleavage, and embryo development of vitrified bovine oocytes. In Experiment 1, the nuclear maturation (MII) rate of immature bovine COCs vitrified was compared between two equilibration times (0 vs 10 min) in vitrification solution 1 (VS1) and two cryodevices (cryotop vs 0.25 mL straw). The MII rate was higher in the non-vitrified control group than in vitrified groups (61 vs 16%, P < 0.0001). Equilibration time did not affect MII rate (P = 0.964); however, the MII rate was higher for COCs vitrified on cryotops than in straws (23 vs 9%, P = 0.007). In Experiment 2, bovine COCs were vitrified on cryotops using two equilibration times (0 vs 5 min) in VS1 and two kinds of vitrification solutions (freshly prepared vs frozen). Cleavage and blastocyst rates were higher (P < 0.0001) in the non-vitrified control group than vitrified groups (cleavage rate 93 vs 42% and blastocysts rate 31 vs 0.4%). Cleavage rate of COCs vitrified using frozen solutions with 5 min equilibration was higher (P = 0.05) than other treatment groups. However, blastocyst rate did not differ (P = 0.993) among treatment groups. In conclusion, cryotop was a better cryodevice than 0.25 mL straw for vitrification of bovine COCs. Furthermore, 5 min equilibration in VS1 improved cleavage. Compared with control, the vitrification procedure per se damaged bovine COCs, resulting in poor nuclear maturation and embryo development. However, vitrification did not immediately kill oocytes, as the cleavage rate was acceptable.
Theriogenology 03/2011; 75(4):602-9. · 1.96 Impact Factor
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ABSTRACT: Despite the importance of cryoprotectants for avoiding ice crystal formation, the high concentrations required for vitrification may be toxic to bovine oocytes. During warming (thawing), the removal of permeating cryoprotectants from cells can lead to osmotic injury, and the most appropriate time interval for warming and cryoprotectant removal from vitrified oocytes is currently uncertain. The present study aimed to evaluate the effect of cryoprotectant exposure, vitrification, and warming time of bovine cumulus oocyte complexes (COC) on fertilization and ability to develop as embryos in vitro. Follicles <8mm in diameter were aspirated from slaughterhouse-derived bovine ovaries. Cumulus oocyte complexes with ≥3 layers of cumulus cells and a uniform cytoplasm were selected, washed 3 times in Dulbecco's PBS+5% newborn calf serum (CS), and randomly divided into 4 groups: 1) control group: no treatment; 2) VS1 group: COC were exposed to vitrification solution 1 [VS1: 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) in TCM-199+20% CS] for 5min; 3) VS1+VS2 group: COC were exposed to VS1 for 5min followed by vitrification solution 2 (VS2: 15% EG, 15% DMSO, and 0.5M sucrose in TCM-199+20% CS) for 30s; and 4) vitrified group: COC were exposed to VS1 and VS2, and then vitrified in liquid nitrogen using cryotops. The COC in VS1, VS1+VS2, and vitrified groups were exposed to a warming solution (0.5M sucrose in TCM-199) for 1 or 5min. The COC from all groups were in vitro matured (IVM) for 22h in TCM-199 containing 5% CS, 5μgmL(-1) LH, 0.5μgmL(-1) FSH, and 0.05μgmL(-1) gentamicin at 38.5°C, 5% CO(2), and high humidity, incubated with frozen-thawed sperm in Brackett-Oliphant capacitating medium for 18h, and the presumptive zygotes were cultured in Charles Rosenkrans 1 amino acids (CR1aa)+5% CS for 9 days. Data were analysed using Proc Glimmix in SAS(®) 9.2 (SAS Institute Inc., Cary, NC, USA). Cleavage and blastocyst rates in the vitrified group (25 and 2%, respectively) were significantly lower (P<0.0001) than in control (75 and 27%), VS1 (68 and 19%), or VS1+VS2 (63 and 22%) groups. Cleavage and blastocyst rates did not differ among non-vitrified groups (P>0.05). In VS1, VS1+VS2, and vitrified groups, warming time had no effect on cleavage or blastocyst rates (P>0.05). In conclusion, although cryoprotectant exposure and warming times had no apparent adverse effect, vitrification of bovine COC drastically reduced cleavage and blastocyst rates. Further studies are required to understand how vitrification of bovine COC affects subsequent fertilization and embryo development.
Reproduction Fertility and Development 01/2011; 23(1):148. · 2.11 Impact Factor
