2012 49 6 1158-1164 Xinjiang Agricultural Sciences doi 10. 6048 /j. issn. 1001-4330. 2012. 06. 027 1 2 3 1 3 1 3 4 5 5 1 3 1. 810001 2. 100049 3. 810001 4. 810007 5. 719000 5 20 ~ 25 26 ~ 30 P = 0. 021 20 ~ 25 26 ~ 30 P = 0. 001 20 ~ 25 26 ~ 30 35 mm P = 0. 65 20 ~ 25 35 mm S857. 2 A 1001-4330 2012 06-1158 - 07 Influence of Different Ovary Storage Temperature on Oocyte in Vitro Maturation and Discovery of Yak Oocyte Maturation Cultivation Methods PANG Bo 1 2 3 ZHAO Xin - quan 1 3 GUO Zhi - lin 1 3 4 QU Lei 5 YU Hong - hao 5 GUO Song - chang 1 3 1. Northwest Institute of Plateau Biology Chinese Academy of Sciences Xining 810001 China 2. Graduate University of Chinese Academy of Sciences Beijing 100049 China 3. Key Laboratory of Adaptation and Evolution of Plateau Biota AEPB Northwest Institute of Plateau Biology Chinese Academy of Sciences Xining 810001 China 4. College of Chemistry and Life Science Qinghai University for Nationalities Xining 810007 China 5. Life Science Research Center Yulin University Yulin Qinghai 719000 China Abstract Objective To explore the influence of different ovary storage temperature on oocyte in vitro maturation and the influence of two cultivation methods on yak oocyte maturation. Method The influence of two different ovary storage temperatures on the maturation rate of five kinds of oocytes was compared by means of in vitro maturation of sheep goat yak Mongolia beef and dairy cow oocytes. Besides two cultivation methods of yak oocyte were carried out to compare the influence on their maturation rate. Result As ovary storage temperature was at 20-25 in vitro maturation rate of goat oocyte was significantly higher than that 2012-01 - 14 KSCX2 - YW - Z - 0951 1984 - E - mail pangbo926@ sina. com 1976 - E - mail guo@ nwipb. cas. cn
6 1159 at 26-30 P = 0. 021. When ovary was kept at 20 to 25 the Mongolia beef oocyte maturation rate was significantly higher than that at 26-30 P = 0. 001. When ovary storage temperature was at 20 to 25 the oocyte in vitro maturation rate of sheep yak and dairy cow were higher than those at 26 to 30. In addition the maturation rate of yak oocyte cultivated in four - well plate and 35 mm culture dish showed no significant difference P = 0. 65. Conclusion Sheep goat yak Mongolia beef and dairy cow ovary are advisable to be stored at 20 to 25 which can improve the oocyte in vitro maturation rate. The maturation rate of yak oocyte cultivated in four - well plate is higher than those cultivated in 35 mm culture dish. Key words yak storage temperature cultivation method in vitro maturation oocyte 0 1 2 3 4 5 6 7 8 9 10 11 12 56. 7% 13 199 Tisse culture medium 199 TCM199 Ham's F12 79. 4% 76. 3% 14 TCM199 Fetalcalf serum FCS Follicle stimulating hormone FSH Luteinizing hormone LH 17β Oestradiol_17β 17β_E2 92. 5% 10 ph 5 1 1. 1 3 h 1. 2
1160 49 1. 2. 1 3 h 3 20 ml 2 ~ 8 mm D - PBS + 3% NBS - - 1. 2. 2 2 38. 5 5% CO 2 24 h 1. 2. 3 5 3 h 20 ~ 25 26 ~ 30 5 t 1. 2. 4 20 ~ 25 1. 2. 1 1 120 4 4 0. 5 ml 2 120 2 ml 35 mm t 2 2. 1 20 ~ 25 26 ~ 30 55. 71% ± 2. 5% VS 43. 27% ± 6% 66. 61% ± 1. 2% VS 62. 35% ± 0. 7% 67. 94% ± 4. 1% VS 60. 51% ± 11. 8% 85. 07% ± 2. 2% VS 68. 04% ± 2. 3% 69. 43% ± 1. 8% VS 63. 8% ± 3. 4% 20 ~ 25 26 ~ 30 P = 0. 021 20 ~ 25 26 ~ 30 P = 0. 001 20 ~ 25 26 ~ 30 20 ~ 25 1 21 2
6 1161 Table 1 1 Oocyte number per ovary of sheep goat yak Mongolia beef and diary cow Ovary number Oocyte number Oocyte number per ovary Sheep 1 127 3 443 3. 05 Goat 536 1 646 3. 07 Yak 257 1 177 4. 58 Mongolia beef 808 3 066 3. 79 Dairy cow 722 3 694 5. 11 Table 2 2 Influence of ovary preserved temperature to in vitro mature rate of oocytes Sheep Goat Yak Mongolia beef Dairy cow 20 ~ 25 0. 557 ± 0. 055 8 ns 0. 666 ± 0. 028 8 * 0. 679 ± 0. 092 5 ns 0. 851 ± 0. 051 2 ** 0. 694 ± 0. 039 4 ns 26 ~ 30 0. 465 ± 0. 109 0. 624 ± 0. 016 6 0. 725 ± 0. 041 2 0. 680 ± 0. 053 5 0. 638 ± 0. 077 3 ns * P < 0. 05 ** P < 0. 01 Note ns indicates no significance * indicates P < 0. 05 ** indicates P < 0. 01 ns * P < 0. 05 ** P < 0. 01 Note ns indicates no significance * indicates P < 0. 05 ** indicates P < 0. 01 numbers above the bars show the number of samples. 1 Fig. 1 Influence of ovary preserved temperature to in vitro mature rate of oocytes 2. 2 64. 01% ± 10. 18% 35 mm 58. 04% ± 7. 68% P = 0. 65 35 mm 3 3
1162 49 Table 3 3 Maturation rate of yak oocyte with two cultivation methods Yak 4 - well plate 0. 640 ± 0. 249 ns 35mm 35mm culture dish 0. 580 ± 0. 188 ns Note ns indicates no significance Fig. 2 2 Oocytes of yak after in vitro mature culture mrna 15 16 30 37 ~ 39 8 h 25 8 h 17 Moodie 22 28 ~ 32 22 ~ 24 35 ~ 37 M I M I M II 22 ~ 24 4 ~ 5 h 35 ~ 37 20 ~ 25 26 ~ 30 20 ~ 25 26 ~ 30
6 1163 18 19 20 21 22 23 4 4 4 90% 95% 30% 67. 94% 85. 07% 69. 43% 90% 60% 15 55. 71% 66. 61% 16 24 4 20 ~ 25 26 ~ 30 55. 71% ± 2. 5% VS 43. 27% ± 6% 66. 61% ± 1. 2% VS 62. 35% ± 0. 7% 67. 94% ± 4. 1% VS 60. 51% ± 11. 8% 85. 07% ± 2. 2% VS 68. 04% ± 2. 3% 69. 43% ± 1. 8% VS 63. 8% ± 3. 4% 20 ~ 25 26 ~ 30 P = 0. 021 20 ~ 25 26 ~ 30 P = 0. 001 20 ~ 25 26 ~ 30 20 ~ 25 64. 01% ± 10. 18% 35 mm 58. 04% ± 7. 68% P = 0. 65 35 mm 1 Cho W Stern S Biggers J. Inhibitory effect of dibutyryl CAMP on mouse oocyte maturation in vitro J. Journal of Experimental Zoology 2005 187 383-386. 2 Wilmut I Paterson L. Somatic cell nuclear transfer J. Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics 2003 136 303-307. 3 Morgan P Warikoo P Bavister B. In vitro maturation of ovary oocytes from unstimulated rhesus monkeys assessment of cytoplasmic maturity by embryonic development after in vitro fertilization J. Biology of Reproduction 1991 45 89-93. 4 Zhang J Wang C Krey L et al. In vitro maturation of human preovulatory oocytes reconstructed by germinal vesicle transfer1 J. Fertility and sterility 1999 71 726-731. 5 Meinecke B Meinecke - Tillmann S. Effects of gonadotropins on oocyte maturation and progesterone production by porcine ovarian follicles cultured in vitro J. Theriogenology 1979 11 351-365. 6 Hewitt D England G. Synthetic oviductal fluid and oviductal cell coculture for canine oocyte maturation in vitro J. Animal reproduction science 1999 55 63-75.
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