Abstract
This study was undertaken to investigate the influence of exposure and removal of four different cryoprotectants (CPAs) on the ATP content of cumulus cell-enclosed (COs) and cumulus cell-denuded (DOs) immature porcine oocytes. The in vitro nuclear maturation of the COs, exposed to and removed from the CPAs was also assessed. Both COs and DOs were exposed to 1.5 M concentrations of each CPA (ethylene glycol (EG); propylene glycol (PG); dimethyl-sulfoxide (DMSO); and glycerol (G)) for durations of 5, 15, and 30 minutes at room temperature (23.5±1.5°C), and immersed in physiological saline supplemented with 20% (v/v) fetal bovine serum for 5 minutes (39°C) to remove each CPA. Before, during and after exposure to each CPA, the ATP content of both the COs and the DOs was measured. After removal from each CPA an aliquot of the COs was matured for 44±2 h, and their nuclear maturation rates were measured up to the metaphase stage of the second meiotic division (the M-II stage). The maturation rates up to the M-II stage were not significantly different between all the groups that were exposed to each CPA for 5 minutes. For 15 and 30 minute exposures, the maturation rates of the COs exposed to PG, DMSO and EG were almost the same as those of the control groups; however, the rates of G group exposed for 15 and 30 minutes were significantly lower (p<0.05) than the control group. These groups were also found to have a decrease in the ATP content of COs and DOs during and after exposure for the same periods (p<0.05). In addition, although the ATP contents of the COs after exposure to EG for any period were the same as the controls, the ATP content of the DOs exposed to EG for any period were significantly lower (p<0.05) than those of the controls. When the ATP content of the COs and DOs of each CPA were compared, the DOs exposed to PG were found to have a significantly greater level (p<0.05) than DOs exposed to G for any duration. In addition, the ATP content of DOs exposed to PG for 30 min and removal was also higher (p<0.05) than when exposed to DMSO for the same period. These findings indicate that PG may be a useful CPA for the cryopreservation of immature porcine oocytes.
Highlights
Oocyte cryopreservation provides greater flexibility in breeding programs than embryo cryopreservation (Payner and Fuller, 2007)
When the ATP content of COs and denuded of their cumulus cells (DOs) exposed to each CPA were compared, the DOs exposed to propylene glycol (PG) were found to have high levels both during and after exposure for any time period, and they were significantly higher (p
The maturation rate and ATP content of COs and DOs in the carrier solution groups were almost identical to the control
Summary
Oocyte cryopreservation provides greater flexibility in breeding programs than embryo cryopreservation (Payner and Fuller, 2007). Aside from our study, on structures such as microtubules (a main component of no reports describe the relationship between CPAs, ATP the meiotic spindle) and microfilaments; this toxicity causes content, or attachment of the cumulus cells to the oocytes; disassembly, which can be reversed if the CPA exposure is we reported that the use of 1.5 M DMSO+0.25 M sucrose of a relatively brief duration or at lower temperatures as a cryoprotectant decreased the ATP content of matured (Fuller, 2004; Gardner et al, 2007). The bovine oocytes in vitro (under the presence or absence of denaturation of proteins in the cytoplasm, degeneration of cumulus cells) (Tsuzuki et al, 2001) After presenting this the cell membrane induced by the high osmolarity, report, we inferred that further study was necessary to relocation of cell organelles and zona hardening of the elucidate the relationship between CPAs, attached cumulus oocytes can be induced by CPAs 2001; Fuller, 2004; Coticchio et al, 2007)
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