山羊卵母细胞冷冻保存及体外受精的研究
摘要
本文主要采用两种不同组合的冷冻液(VSⅠ和VSⅡ)OPS法玻璃化冷冻不同期(GV、10h、IVM)的山羊卵母细胞,并对冷冻后的卵母细胞进行体外受精实验。主要结果如下:
1.用两种不同组合的冷冻液VSⅠ(EG+DMSO)和VSⅡ(PROH+DMSO)冷冻各期(GV、10h、IVM)山羊卵母细胞。经用VSⅠ冷冻液冷冻后,GV期的形态正常率、成熟率、受精率分别为86.2%、25.3%、4.0%;培养10h后的分别为86.7%、25.6%、5.1%;IVM期的冻后形态正常率为87.8%、受精率为13.9%。经用VSⅡ冷冻液冷冻后GV期的形态正常率、成熟率、受精率分别86.9%、23.3%、2.7%;培养10h后的分别为88.0%、24.5%、3.3%;IVM期的冻后形态正常率为86.0%、受精率为13.5%。两组冷冻液对应各期的冻后形态正常率、成熟率和受精率差异均不显著(P>0.05)。
2.无论是VSⅠ(EG+DMSO)还是VSⅡ(PROH+DMSO),山羊卵母细胞在GV期、体外培养10h后以及IVM期的冻后形态正常率上差异均不显著(P>0.05);GV期和体外培养10h后的卵母细胞的冻后成熟率差异不显著(P>0.05);但GV期和体外培养10h后卵母细胞的冻后受精率均低于IVM期卵母细胞,差异显著(P<0.05)。
3.用获能精子和未获能精子,对成熟卵母细胞进行显微授精后,两组的形态正常率分别为81.5%和82.7%,差异不显著(P>0.05),获能精子组的受精率(28.3%)比未获能的精子组(18.8%)高,但差异不显著(P>0.05)。
4.常规体外受精(IVF)的受精率(13.9%)比显微授精(ICSI)(28.3%)的低,两者差异显著(P<0.05)。说明ICSI技术相比较常规IVF更能够提高卵母细胞的受精率。
In this paper, different stage of goat oocytes(GV、10h、IVM) were vitrified with two different concentration freezing media (VSⅠand VSⅡ) with the OPS method, after vitrified those oocytes were fertilized with IVF or ICSI. The main results were as follows:
1. Vitrified different stages of goat oocytes(GV、10h、IVM) with two different concentration freezing media of VSⅠ(EG+DMSO)and VSⅡ(PROH+DMSO). After vitrified with VSⅠ, the morphologically normal rates、the IVM rates and the fertility rate of GV stage were 86.2%、25.3%、4.0%,respectively; and cultured 10h in vitro were 86.7%、25.6%、5.1%,respectively; the morphologically normal rate and the fertility rate of IVM stage were 86.0% and 13.5%, respectively. After vitrified, there was no significant difference in the morphologically normal rates、the IVM rates or the fertility rate between VSⅠ(EG+ DMSO) and VSⅡ( PROH+ DMSO) (P>0.05) .
2. Either VSⅠ( EG+ DMSO) or VSⅡ( PROH+ DMSO), there was no significant difference in the morphologically normal rates、the IVM rates or the fertility rate of goat oocytes between each other of GV stage ,cultured 10h in vitro and IVM stage (P>0.05); there showed no significant difference between the IVM rates of GV stage and developed 10h in vitro; but the fertility rate of oocytes of GV stage and developed 10h in vitro were both lower than IVM, the difference was significance ( P<0.05).
3. After microinsemination with capacitation and incapacitation sperms, the morphologically normal rates of maturate oocytes were 81.5%and 82.7%, respectively,
1.用两种不同组合的冷冻液VSⅠ(EG+DMSO)和VSⅡ(PROH+DMSO)冷冻各期(GV、10h、IVM)山羊卵母细胞。经用VSⅠ冷冻液冷冻后,GV期的形态正常率、成熟率、受精率分别为86.2%、25.3%、4.0%;培养10h后的分别为86.7%、25.6%、5.1%;IVM期的冻后形态正常率为87.8%、受精率为13.9%。经用VSⅡ冷冻液冷冻后GV期的形态正常率、成熟率、受精率分别86.9%、23.3%、2.7%;培养10h后的分别为88.0%、24.5%、3.3%;IVM期的冻后形态正常率为86.0%、受精率为13.5%。两组冷冻液对应各期的冻后形态正常率、成熟率和受精率差异均不显著(P>0.05)。
2.无论是VSⅠ(EG+DMSO)还是VSⅡ(PROH+DMSO),山羊卵母细胞在GV期、体外培养10h后以及IVM期的冻后形态正常率上差异均不显著(P>0.05);GV期和体外培养10h后的卵母细胞的冻后成熟率差异不显著(P>0.05);但GV期和体外培养10h后卵母细胞的冻后受精率均低于IVM期卵母细胞,差异显著(P<0.05)。
3.用获能精子和未获能精子,对成熟卵母细胞进行显微授精后,两组的形态正常率分别为81.5%和82.7%,差异不显著(P>0.05),获能精子组的受精率(28.3%)比未获能的精子组(18.8%)高,但差异不显著(P>0.05)。
4.常规体外受精(IVF)的受精率(13.9%)比显微授精(ICSI)(28.3%)的低,两者差异显著(P<0.05)。说明ICSI技术相比较常规IVF更能够提高卵母细胞的受精率。
In this paper, different stage of goat oocytes(GV、10h、IVM) were vitrified with two different concentration freezing media (VSⅠand VSⅡ) with the OPS method, after vitrified those oocytes were fertilized with IVF or ICSI. The main results were as follows:
1. Vitrified different stages of goat oocytes(GV、10h、IVM) with two different concentration freezing media of VSⅠ(EG+DMSO)and VSⅡ(PROH+DMSO). After vitrified with VSⅠ, the morphologically normal rates、the IVM rates and the fertility rate of GV stage were 86.2%、25.3%、4.0%,respectively; and cultured 10h in vitro were 86.7%、25.6%、5.1%,respectively; the morphologically normal rate and the fertility rate of IVM stage were 86.0% and 13.5%, respectively. After vitrified, there was no significant difference in the morphologically normal rates、the IVM rates or the fertility rate between VSⅠ(EG+ DMSO) and VSⅡ( PROH+ DMSO) (P>0.05) .
2. Either VSⅠ( EG+ DMSO) or VSⅡ( PROH+ DMSO), there was no significant difference in the morphologically normal rates、the IVM rates or the fertility rate of goat oocytes between each other of GV stage ,cultured 10h in vitro and IVM stage (P>0.05); there showed no significant difference between the IVM rates of GV stage and developed 10h in vitro; but the fertility rate of oocytes of GV stage and developed 10h in vitro were both lower than IVM, the difference was significance ( P<0.05).
3. After microinsemination with capacitation and incapacitation sperms, the morphologically normal rates of maturate oocytes were 81.5%and 82.7%, respectively,
引文
1 中国农业大学主编. 家畜繁殖学,北京:中国农业出版社,2000.
2 中国农业大学主编. 遗传学,北京:中国农业出版社,2000.
3 陈荪,吴明章. 卵母细胞和卵巢组织的冷冻. 生殖医学杂志, 2001, 10(6):374~378.
4 陈大元. 受精生物学,北京:科学出版社,1999, 154~155.
5 Sherman JK, Lin. Survival of unfertilized mouse eggs during freezing and thawing. Proc Soc Exp Biol Med,1958, 98:902-905.
6 Whittingham DG. Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at –196℃. Reprod Fertil,1977, 49:89-94.
7 Leibo SP. Water permeability and its activation energy of fertilized and unfertilized mouse ova. Membr Biol,1980, 53:179-188.
8 Glenister PH, Wood MJ, Kirby C, et al.. Incidence of chromosome anomalies in first-cleavage mouse embryos obtained from frozen-thawed oocytes fertilized in vitro. Gamete Res,1987, 16:205-216.
9 Karlsson JOM, ErogluA, Toth TL, et al.. Fertilization and development of mouse oocytes cryopreserved using a theoretically optimized protocol. Hum Reprod, 1996, 11:1296-130.
10 Nakagata N. High Survival Rate of Unfertilized Mouse Oocytes After vitrification. J Reprod Fert,1989,87:479-483.
11 Van Blerkom J, Davis PW. Cytogenic, cellular, and developmental consequences of cryopreservation of immature and mature mouse and human oocytes. Microsc Res Tech, 1994, 27: 165~193.
12 Al-Hassanis S, Kirsch J, Diedrich K, et al.. Successful embryo transfer of cryo- preserved and in-vitro fertilized rabb it oocytes. Hum Reprod, 1989, 4:77-79.
13 Lim J M, Fukui Y, Ono H, Developmental competence of bovine oocytes frozen at various maturation stages followed by in vitro maturation and fertilizationTheriogenology, 1992, 37:351-361.
14 Fuku E, Kojima T, Shioya Y, et al.. Downey BR. In vitro fertilization and development of frozen-thawed bovine oocytes. Cryobiology, 1992, 9:485-492.
15 Chen C. Pregnancy after human oocyte cryopreservation. Lancet, 1986, 1: 884- 886.
16 Van Uem J, Siebzehnrubl ER, Schuh B, et al.. Birth after cryopreservation of unfertilized oocytes. Lancet, 1987, (1): 752-753.
17 Mandelbaum J. Cryopreservation in human assisted reproduction is now routine for embryos but remain a research procedure for oocytes. Hum Reprod, 1998, 13 (3):161-174.
18 Tucker MJ. Birth after cryopreservation of immature oocytes with subsequent with subsequent in vitro maturation.FertilSteril.1998, 70:578-579.
19 Yoon TK, Chung HM, Lim JM, et al.. Pregnancy and delivery of healthy infants developed from vitrified oocytes in a stimulated in vitro fertilization embryo transfer program. Fertile Sterol, 2000, 74:180-181.
20 Aman RR, Parks JE. Effects of cooling and rewarming on the meiotic spindle and chromosomes of in vitro-matured bovine oocytes. Bio Reprod, 1994; 50:103-110.
21 Albertini DF. Regulation of meiotic maturation in the mammalian oocyte: Interplay between exogenous cues and the microtubule cytoskeleton. Bio Essays.1992, 1:97-103.
22 Eroglu A, Toth TL, Toner M. Cytoskeletal organization of cryopreserved mouse oocytes during in vitro maturation and fertilization.Cryobiology.1997, 5:351.
23 Ashwood-Smith MJ, Morris GW, Fowler R, et al.. physical factors are involved in the destruction of embryos and oocytes during freezing and thawing procedures. Hum Ropod.1998, 3795-802.
24 Vincent C ,Pickering SJ, Johnson MH.The hardening effect of dimethylsulphoxide on the mouse zona pellucida require the presence of an oocyte and is associatedwith a reduction in the number of cortical granules present. J Reprod Fertil, 1990, 89: 253-259.
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32 Galf LE, Baril G, Vallet JC, et al.. In vivo and in vitro survival of goat embryos after freezing with ethylene glycol or glycekol. Theriogenology,1993,40:771-777.
33 Miyake T, Kassi M, Zhu S E, et al.. Vitrification of mouse oocytes and embryos at various stages of development in an ethylene glycol-based solution by a simple method. Theriogenology, 1993, 40:121-134.
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2 中国农业大学主编. 遗传学,北京:中国农业出版社,2000.
3 陈荪,吴明章. 卵母细胞和卵巢组织的冷冻. 生殖医学杂志, 2001, 10(6):374~378.
4 陈大元. 受精生物学,北京:科学出版社,1999, 154~155.
5 Sherman JK, Lin. Survival of unfertilized mouse eggs during freezing and thawing. Proc Soc Exp Biol Med,1958, 98:902-905.
6 Whittingham DG. Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at –196℃. Reprod Fertil,1977, 49:89-94.
7 Leibo SP. Water permeability and its activation energy of fertilized and unfertilized mouse ova. Membr Biol,1980, 53:179-188.
8 Glenister PH, Wood MJ, Kirby C, et al.. Incidence of chromosome anomalies in first-cleavage mouse embryos obtained from frozen-thawed oocytes fertilized in vitro. Gamete Res,1987, 16:205-216.
9 Karlsson JOM, ErogluA, Toth TL, et al.. Fertilization and development of mouse oocytes cryopreserved using a theoretically optimized protocol. Hum Reprod, 1996, 11:1296-130.
10 Nakagata N. High Survival Rate of Unfertilized Mouse Oocytes After vitrification. J Reprod Fert,1989,87:479-483.
11 Van Blerkom J, Davis PW. Cytogenic, cellular, and developmental consequences of cryopreservation of immature and mature mouse and human oocytes. Microsc Res Tech, 1994, 27: 165~193.
12 Al-Hassanis S, Kirsch J, Diedrich K, et al.. Successful embryo transfer of cryo- preserved and in-vitro fertilized rabb it oocytes. Hum Reprod, 1989, 4:77-79.
13 Lim J M, Fukui Y, Ono H, Developmental competence of bovine oocytes frozen at various maturation stages followed by in vitro maturation and fertilizationTheriogenology, 1992, 37:351-361.
14 Fuku E, Kojima T, Shioya Y, et al.. Downey BR. In vitro fertilization and development of frozen-thawed bovine oocytes. Cryobiology, 1992, 9:485-492.
15 Chen C. Pregnancy after human oocyte cryopreservation. Lancet, 1986, 1: 884- 886.
16 Van Uem J, Siebzehnrubl ER, Schuh B, et al.. Birth after cryopreservation of unfertilized oocytes. Lancet, 1987, (1): 752-753.
17 Mandelbaum J. Cryopreservation in human assisted reproduction is now routine for embryos but remain a research procedure for oocytes. Hum Reprod, 1998, 13 (3):161-174.
18 Tucker MJ. Birth after cryopreservation of immature oocytes with subsequent with subsequent in vitro maturation.FertilSteril.1998, 70:578-579.
19 Yoon TK, Chung HM, Lim JM, et al.. Pregnancy and delivery of healthy infants developed from vitrified oocytes in a stimulated in vitro fertilization embryo transfer program. Fertile Sterol, 2000, 74:180-181.
20 Aman RR, Parks JE. Effects of cooling and rewarming on the meiotic spindle and chromosomes of in vitro-matured bovine oocytes. Bio Reprod, 1994; 50:103-110.
21 Albertini DF. Regulation of meiotic maturation in the mammalian oocyte: Interplay between exogenous cues and the microtubule cytoskeleton. Bio Essays.1992, 1:97-103.
22 Eroglu A, Toth TL, Toner M. Cytoskeletal organization of cryopreserved mouse oocytes during in vitro maturation and fertilization.Cryobiology.1997, 5:351.
23 Ashwood-Smith MJ, Morris GW, Fowler R, et al.. physical factors are involved in the destruction of embryos and oocytes during freezing and thawing procedures. Hum Ropod.1998, 3795-802.
24 Vincent C ,Pickering SJ, Johnson MH.The hardening effect of dimethylsulphoxide on the mouse zona pellucida require the presence of an oocyte and is associatedwith a reduction in the number of cortical granules present. J Reprod Fertil, 1990, 89: 253-259.
25 George MA, Johnson MH. Use of fetal bovine serum substitutes for the protection of the mouse zona pellucida against hardening during cryoprotectant addition. Hum Reprod.1993, 8:1898-1900.
26 Bouquet M, Selva J, Auroux M. The incidence of chromosomal abnormalities in frozen-thawed mouse oocytes after in-vitro fertilization. Hum Reprod.1992, 7:76-80.
27 Ben-Yosef D, Oron Y, Shalgi R. Low temperature and fertilization-induced Ca~(2+) changes in rat eggs. Mol Repord, 1995, 42:122-129.
28 Jame J, Stachecki, Steen M, et al.. Cryoprreservation of mouse oocytes using a medium with low sodium content: effect of plunge temperature. Cryobiology, 2000, 40(1):4-12.
29 Martinez AG, Matkovic M. Cryopreservation of bovine embryos: slow freezing and vitrification. Theriogenology, 1998, 49(5):10391049.
30 Scheffen B, Van Der Zwalmen P, Massip A. A simple and efficient procedure for preservation of mouse embryos by vitrification. Cryo-letters, 1986, 7:260-269.
31 Suzuki T, Takagi M, Yamamoto M, et al.. Pregnancy rate and survival in culture of in vitro fertilized bovine embryos frozen in various cryoprotectants and thawed using a one-step system.Theriogenoiogy,1993,40:651-659.
32 Galf LE, Baril G, Vallet JC, et al.. In vivo and in vitro survival of goat embryos after freezing with ethylene glycol or glycekol. Theriogenology,1993,40:771-777.
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