| The oocytes activation is one of the key step of nuclear transfer(NT) in mammal, and the efficiency of oocytes activation affects the efficiency of NT directly. Nowadays the efficiency of NT in mammal is very low, one reason is that the oocytes are not activated enough. Therefore it is very necessary to research the oocytes activation.By now, many mammalian oocytes activation have been researched, but the study of pig oocytes activation is not enough.This study was undertaken to explore the methods ,such as electric-activation, ionomycin , 6-dimethylamin-opurine(6-DMAP), cytochalasin B(CB) for activation of the in-vitro matured porcine oocytes, and the effect on development after activation, in order to obtain an optimal method for pig in vitro-matured oocytes.The porcine oocytes matured for 44~48 hours in vitro were treated with different intensity of direct current field strengths (1.0 KVcm~-1,1.3 KV cm~-1,1.5 KV cm~-1, 2.0KVcm~-1), pulse duration (40μs, 80μs, 100μs, 150μs) , pulse times(l time,2 times,3 times). The results were as following: (l)The activation rates and development rates of the oocytes altered with the electric field strength, pulse duration and times. When pulse duration and times were fixed, the percentage of activation and cleavage in 1.3 KV cm~-1 electric strength group was respectively significantly higher than those in 1.0KV cm~-1 electric strength group (68.75%vs44.38%; 74.54%vs50.70%, P<0.01) . Electric strength was raised again, but the activation percentage and cleavage percentage started to decrease. The percentage of activation and cleavage in either 1.5 KV cm~-1 electric strength group or 2.0KV cm~-1 electric strength group was respectively significantly lower than those in 1.3 KV icm "'electric strength group(56. 25%, 64. 44%vs68.75%; 54. 38%, 60. 92%vs74.54%, P < 0. 05). (2)When the electric strength was fixed in 1.3 KV cm~-1, one time pulse, when pulse durantion was 80μs, the percentage of activation and cleavage was high to 70.63% and 75. 22%, it was significantly higher than that when the pulse durantion was 40us(50.63%, 53.09%; P < 0.05) . Pulse duration raised continuely, the activation percentage and cleavage percentage did not raise. When pulse duration was 100μs or 150μs, the percentage of activation and leavage was respectively significantly lower than pulse durantion was 80μs group(55.63%, 67.42%vs70.63%; 53.13%, 57.65%vs75.22%, P < 0.05) . (3)On condition that 1.3 KV cm~-1 for 80ns, the percentage of activation and cleavage in one time pulse group was respectively significantly higher than those in two times and three times(71.25%vs56.88%, 53.75%; 75.44%vs60.44%, 56.98%, P < 0.01). The result showed that one pulse was enough to activate pig oocytes.The porcine oocytes matured for 44~48 hours in vitro were treated with ionomycin indifferent concentration(l5μmol·L~-1, 20μmol·L~-1, 25μmol·L~-1) for different time (20min, 30 min, 40 min, 50 min) . The results were as following: The oocytes treated with ionomycin in different concentration(15μmol·L~-1. 20μmol·L~-1 25μmol·L~-1) for 40 minutes, the activation rates and cleavage rates were 57.50%, 59.78%; 60.63%, 62.89%; 58.75%, 60.64% respectively. Although the percentage of activation and cleavage in the group that 20μmol·L~-1 was the highest, but compare with the others there was no significant difference among them (P > 0.05) . The oocytes treated with 20μmol·L~-1 ionomycin for different time (20min, 30 min, 40min,50min) , the activation rates and cleavage rates were 56. 88%, 57.14%; 58.75%, 58.51%; 61.25%, 61.22%; 59.38%, 58. 95% respectively. Although the percentage of activation and cleavage in the group that 40 minutes was the highest, but compare with the others there was no significant difference between them (P > 0.05 ). The result showed that the concentration and time treated with ionomycin did not significantly effect the activation rate of porcine oocyte.The porcine oocytes matured for 44~48 hours in vitro were respectively treated with different electric pulse(1.5 KV · cm~-1, 40us, 1 time; 1.3 KV · cm~-1, 80us , 1 time; 1.0 KV · cm~-1 120μs, 1 time) , then cultured in the NCSU23 containing 6-DMAP and CB for 4 hours. The results were as following: When oocytes were first activated by electricity and then cultured in the NCSU23 containing 6-DMAP and CB, the percentage of activation and cleavage was high to 74.38%, 70.59%; 81.88%, 84.73%; 70.63%, 68.14%, it was significantly higher than the control group(64.38%, 62.14%; 70.00%, 76.79%; 60.63%, 57.73%, P < 0.05).The porcine oocytes matured for 44-48 hours in vitro were respectively treated with ionomycin (15μmol·L~-1, 50 min; 20μmol·L~-1, 40 min; 25μmol·L~-1, 30 min), then cultured in the NCSU23 containing 6-DMAP and CB for 4 hours. The results were as following: When oocytes were first activated by ionomycin and then cultured in the NCSU23 containing 6-DMAP and CB, the percentage of activation and cleavage was high to 63.13%, 62.38%; 68.13%, 68.87%; 65.00%, 64.42%, it was significantly higher than the control group(57.50%, 57.61%; 61.88%, 61.62%; 58.13%, 58.06%, P< 0.05).The porcine oocytes matured for 44-48 hours in vitro were activated by different methods of parthenogenetic activation, A: electric-activation+6-DMAP+CB; B: ionomycin+6-DMAP+ CB; C: electric-activation+ ionomycin+6-DMAP+CB. The results were as following: The percentage of activation and cleavage of group A was respectively in 81.25% and,it was significantly higher than group B(69.38%, 70.27%; P < 0.01) . The percentage of activation of group C was in 86. 88%, there was no significant difference between group A. The percentage of cleavage was high to 89. 21%, it is significantly higher than group A (83.25%, P< 0.05).In conclusion, the optimal electric stimulation conditions of porcine oocytes matured in vitro for 44-48 hours is 1.3 KV · cm~-1 direct current field strength, 80us pulse duration,one time pulse; that in the concentration of 15~25μmol·L~-1 and in the time of 20~50 min treated with ionomycin... |
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