| Germial vesicle (GV) oocyte cyropreservation is of the great importance, but ithasn't reported systematically by now. The objective of this study was to establish aneffective method to cryopreserve bovine GV oocytes. This work will be helpful forthe biotechnological research and practice, such as in vitro fertilization, nucleartransfer and transgenic technique. The present study investigated the effects ofpermeated cryoprotectants, pretreatment methods, cryoprotective procedures, oocytesource from different size follicles. The results as follows: When GV bovince oocytes was cryopreserved through programmed procedure,1.5 M Ethylene glycol (EG), 1.5 M 1, 2-propanediol (PROH), and 0.75 M EG plus0.75 M PROH was added to 0.1 M sucrose and 10% FCS as cryoprectantsrespectively, bovine GV oocytes maturation rate were 10.7%, 9.8%, 7.9%,respectively after in vitro culture, and no significant difference was found amongthem (P>0.05). Thereafter, 1.5 M EG was used as penetreated cryopresertectant, 0(control), 0.1, 0.2, 0.3 M sucrose, 10%, 20%, 30% FCS was supplementedrespectively. The result showed that the maturation rates had no significant differenceafter cryopreservation when any single supplementation (P>0.05). Whereas both 0.1M sucrose and 20% FCS were added in 1.5 M EG, the maturation rate wassignificantly higher than the control (14.3% vs 5.9%;P<0.05). In order to evaluate the toxicity of the vitrification solutions, the oocytes weretreated in FS (30% ficoll and 0.5 M sucrose) or EFS20 (FS: EG: FCS=7: 2: 1) 5, 10,15, 20 min, or EFS40 (FS: EG: FCS=5: 4: 1) 1, 3, 5, 10 min respectively. The oocyteswere treated in DPBS 20 min as control. The results showed that the maturation rateof the oocytes treated in FS more than 15 min was significantly lower than the control(P<0.05). When the oocytes were treated with EFS20 more than 10 min, thematuration rates was most significantly lower than the control (P<0.01). When withEFS40 1 min, the maturation rate significantly reduced, and most significantlyreduced when the oocytes were treated more than 3 min. The results indicate that FShas no toxicity towards oocytes when treating time was short. The toxicity of EFS20and EFS40 enhanced rapidly when time increased. All in all, the toxicity ofvitrification solution increased along with the EG concentration increase and treatingtime. Bovine GV oocytes were cryopreserved by the glass micro-pipe (GMP) or the straw as carrier with different pretreatment methods, respectively. The results showed that the maturation rate of two step had no difference with one step no matter GMP or Straw was used (GMP, 25.0% vs 22.1%, P>0.05; Straw, 11.6% vs 8.1%, P>0.05). The maturation rate of GMP was significantly higher than that of Straw (P<0.05). When bovine GV oocytes was vitrified by two step method through GMP (glassmicropipe), the oocytes were pretreated with EFS20 0.5, 1, 2 and 3 min, respectively,and then treated with EFS40 30 s. The results showed that the maturation rates were9.3%, 10.3%, 25.0% and 12.5%, respectively. Among them, the maturation rate of 2min was significantly higher than the others (P<0.05), and there were no differencesbetween others (P>0.05). Oocytes were treated in EFS40 1, 2, 3, 5 min by one stepmethod respectively, the maturation rates of 2 and 3 min (16.1%, 22.1%) weresignificantly higher than the others (0.1%, 3.1%). The maturation rates between 2 and3, and between 1 and 5 min had no difference (P>0.05). In view of highest maturationrate, the two methods had the similar effect (25.0% vs 22.1%, P>0.05). Based on the previous results, GMP was chosen to cryopreserve the oocytescollected from different size of follicle (2-6mm and <2mm in diameter). There was nodifference of maturation rate between them (27.1% vs 21.6%, P>0.05). In conclusion, the supplementation of both 0.1 M sucrose and 20% FCS canimprove the maturation rate when bovine GV oocytes were programme-cryopreserved.For vitrified bovine GV oocytes, two step method (EFS20 2 min, EFS40 30 s) isbetter than one step by GMP. Vitrification is better than programming. GV o...
|
PDF Full Text Request