鹿卵母细胞体外成熟与体外受精的研究
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摘要
随着胚胎生物工程技术的深入研究和不断发展，在我国开展对鹿胚胎体外生产技术的研究显得越来越重要与紧迫。本研究以我国的马鹿为试验对象，对鹿胚胎体外生产体系中最关键的基础性环节——鹿卵母细胞的体外成熟和体外受精进行探索，旨在提出一套在实践中具有可操作性的鹿卵母细胞体外培养体系，进而为鹿体外受精研究奠定基础。试验研究结果表明：
1、采用递减法肌肉注射Folltropin-V(Canada)并结合PG对马鹿进行超数排卵处理可以有效提高母鹿卵巢卵母细胞的采集数量。马鹿在埋入CIDR后第8、9、10、11天内每日注射Folltropin-V(Canada)的剂量分别为：120～140mg、80～100mg、60mg、40mg；或将第四天剂量用PMSG(500IU／头)代替，均可以得到较多的卵母细胞。
2、采用先抽吸再切割的方法可以提高所获取鹿COCs的数量。切割法的补充使用所检得的卵母细胞数约占检取卵母细胞总数的1／2(43枚／84枚)。两种方法的操作时间不超过3min／对卵巢。
3、将所获卵泡液和切割液经过冲洗处理后再检卵与直接检卵相比：视野明了，蛋白质等物质不易发生凝集，卵的移取方便、快速。
4、检取的鹿卵母细胞可分为4级：A级为卵丘细胞致密，不扩散，细胞质均匀，形状规则，至少完全包裹有4层卵丘细胞；B级为卵丘细胞1～3层，基本上包裹卵母细胞；C级为半裸卵，卵母细胞外有部分卵丘细胞存在；D级为裸卵、变形卵，包括退化的卵母细胞。研究结果表明：A、B级卵母细胞体外培养成熟率较高。
5、马鹿的卵母细胞在体外成熟培养中采用37℃和38.5℃两种温度条件的比较结果是：38.5℃条件下的卵母细胞成熟率(78.9％)显著(p＜0.05)高于37.0℃时的卵母细胞成熟率(44.4％)。
6、以M199作为马鹿卵母细胞体外成熟培养的培养基，所配制的成熟液(1)：M199+10％FBS+0.2mM丙酮酸钠+1mM双抗+10mM Hepes+10mg／ml GnRH+1mg／mlE_2与成熟液(2)：M199+20％FBS+0.2mM丙酮酸钠+1mM双抗+10mg／ml GnRH，都适宜进行卵母细胞的体外成熟培养，体外成熟率可以分别达到78.9％、84.2％。
7、采用上浮法可有效地使鹿的精子获能。培养的条件为38.5℃、5％CO_2、饱和湿度；洗精时两次离心的速度分别为2200rpm、2000rpm为宜；TALP沈精／受精液(2)与BO洗精／受精液(2)，处理精子的效果分别好于TALP沈精／受精液(1)与BO洗精／受精液(1)；BO洗精／受精液(2)比TALP沈精／受精液(2)处理马鹿精子的获能效果更佳，精子存活时间可长达24h，后者达到9h。
8、TALP受精液(2)与BO受精液(2)均可用一J几鹿卵母细胞的体外受精。虽然鹿精子在
两种受精液中的存活时间不同，但都可以得到受精卵，受精率分别为66.7%、70.6%，
两种受精液对受精率的影响差异不显著(p>0.05)。
9、鹿的受精卵在M199胚胎培养液中进行体外培养，发育至2一细胞的比例达50%
以上。
Along with the development of embryo engineering technique, it changes more and more urgent and essential to begin to study in vitro embryo production of deer in our country. We studied in vitro maturation and fertilization of Wapiti(Cervus elaphus) ovary oocytes. The aim of the study is to upbuild a set of perfect protocol of in vitro oocytes culture that is feasible to deer in the practice, so as to give theoretic basis, experience and methods to the deep study of deer IVF.
The results of our studies are as followings:
l.Wapiti(Cervus elaphus) superovulation with Folltropin-V from Canada can increase the number of ovary oocytes derived from abattoir material. The program is as follows: After being treated with CIDR device for 8 d, all hinds received a four-day twice-per-day injection of 120~140mg, 80~100mg, 60mg, 40mg, Folltropin-V, or a single injection of 500IU, PMSG in place of Folltropin-V at the fourth day.
2.The number of COCs collected by first aspirated and then deeply cut from the abattoir-derived deer ovaries can be increased. The method of deeply cut can make up for the shortage of single aspiration and have made the number of COCs increase to twice of single aspiration. The total operating time is within 3minutes.
3.Gently washing the follicular fluid, compared with no washing and direct checking oocytes, has more advantage: convenient, the protein difficultly coacervating,'fastly moving ooocytes and saving time.
4.The COCs can be divided into four degrees:grade A is with fully cumulus cells and have four layers of comulus cells at least; grade B have 1-3 layers fully cumulus cells; grade C is hemi-nude egg with sectional cumulus cells; grade D are nude eggs> deformed oocytes and degenerative oocytes. The COCs of grade A and grade B have higher maturation rates.
5.The in vitro maturation rate of oocytes is obviously higher under 38.5℃ than under 37.0'C(p<0.05).
6.Medium 199 is the culture medium of in our experiments.. The following culture systems are all available and can make the in vitro maturation rate reach 78.9%, 84.2%: (l)M199+10%FBS+0.2mM Na-pyruvate+lmM antibiotic+10mg/ml GnRH +10mM Hepes +lmg/ml E2; (2)M199+20%FBS+0.2mM Na-pyruvate+lmM antibiotic+lOmg/ml GnRH.
7.The method of swim-up is effective for Wapiti(Cervus elaphus) sperm washing and capitation in vitro. The two centrifugalizing speeds in washing sperm being 2200rpm, 2000
rpm is best. The effects of operating in TALP sperm washing or fertilization medium(2) and in BO sperm washing or fertilization medium(2) are all better than in TALP sperm washing or fertilization medium(l) and in BO sperm washing or fertilization medium(l). The time of sperm living in vitro can reach to 24 hour when sperm cultured in BO fertilization medium(2), and the time in TALP-fertilization medium(2) is only 9h.
8.In vitro fertilization with BO or TALP can all make the ovum culturing to cleavage stage, and the fertilization rate are 66.7%, 70.6%. The effects of fertilizing in the two mediums are unobviously(p>0.05).
9.The early embryo of Wapiti(Cerv玸 elaphus) developed in embryo culture madium M199. The rate of the cells that have developed to 2-cell can reach to 50%.
1、采用递减法肌肉注射Folltropin-V(Canada)并结合PG对马鹿进行超数排卵处理可以有效提高母鹿卵巢卵母细胞的采集数量。马鹿在埋入CIDR后第8、9、10、11天内每日注射Folltropin-V(Canada)的剂量分别为：120～140mg、80～100mg、60mg、40mg；或将第四天剂量用PMSG(500IU／头)代替，均可以得到较多的卵母细胞。
2、采用先抽吸再切割的方法可以提高所获取鹿COCs的数量。切割法的补充使用所检得的卵母细胞数约占检取卵母细胞总数的1／2(43枚／84枚)。两种方法的操作时间不超过3min／对卵巢。
3、将所获卵泡液和切割液经过冲洗处理后再检卵与直接检卵相比：视野明了，蛋白质等物质不易发生凝集，卵的移取方便、快速。
4、检取的鹿卵母细胞可分为4级：A级为卵丘细胞致密，不扩散，细胞质均匀，形状规则，至少完全包裹有4层卵丘细胞；B级为卵丘细胞1～3层，基本上包裹卵母细胞；C级为半裸卵，卵母细胞外有部分卵丘细胞存在；D级为裸卵、变形卵，包括退化的卵母细胞。研究结果表明：A、B级卵母细胞体外培养成熟率较高。
5、马鹿的卵母细胞在体外成熟培养中采用37℃和38.5℃两种温度条件的比较结果是：38.5℃条件下的卵母细胞成熟率(78.9％)显著(p＜0.05)高于37.0℃时的卵母细胞成熟率(44.4％)。
6、以M199作为马鹿卵母细胞体外成熟培养的培养基，所配制的成熟液(1)：M199+10％FBS+0.2mM丙酮酸钠+1mM双抗+10mM Hepes+10mg／ml GnRH+1mg／mlE_2与成熟液(2)：M199+20％FBS+0.2mM丙酮酸钠+1mM双抗+10mg／ml GnRH，都适宜进行卵母细胞的体外成熟培养，体外成熟率可以分别达到78.9％、84.2％。
7、采用上浮法可有效地使鹿的精子获能。培养的条件为38.5℃、5％CO_2、饱和湿度；洗精时两次离心的速度分别为2200rpm、2000rpm为宜；TALP沈精／受精液(2)与BO洗精／受精液(2)，处理精子的效果分别好于TALP沈精／受精液(1)与BO洗精／受精液(1)；BO洗精／受精液(2)比TALP沈精／受精液(2)处理马鹿精子的获能效果更佳，精子存活时间可长达24h，后者达到9h。
8、TALP受精液(2)与BO受精液(2)均可用一J几鹿卵母细胞的体外受精。虽然鹿精子在
两种受精液中的存活时间不同，但都可以得到受精卵，受精率分别为66.7%、70.6%，
两种受精液对受精率的影响差异不显著(p>0.05)。
9、鹿的受精卵在M199胚胎培养液中进行体外培养，发育至2一细胞的比例达50%
以上。
Along with the development of embryo engineering technique, it changes more and more urgent and essential to begin to study in vitro embryo production of deer in our country. We studied in vitro maturation and fertilization of Wapiti(Cervus elaphus) ovary oocytes. The aim of the study is to upbuild a set of perfect protocol of in vitro oocytes culture that is feasible to deer in the practice, so as to give theoretic basis, experience and methods to the deep study of deer IVF.
The results of our studies are as followings:
l.Wapiti(Cervus elaphus) superovulation with Folltropin-V from Canada can increase the number of ovary oocytes derived from abattoir material. The program is as follows: After being treated with CIDR device for 8 d, all hinds received a four-day twice-per-day injection of 120~140mg, 80~100mg, 60mg, 40mg, Folltropin-V, or a single injection of 500IU, PMSG in place of Folltropin-V at the fourth day.
2.The number of COCs collected by first aspirated and then deeply cut from the abattoir-derived deer ovaries can be increased. The method of deeply cut can make up for the shortage of single aspiration and have made the number of COCs increase to twice of single aspiration. The total operating time is within 3minutes.
3.Gently washing the follicular fluid, compared with no washing and direct checking oocytes, has more advantage: convenient, the protein difficultly coacervating,'fastly moving ooocytes and saving time.
4.The COCs can be divided into four degrees:grade A is with fully cumulus cells and have four layers of comulus cells at least; grade B have 1-3 layers fully cumulus cells; grade C is hemi-nude egg with sectional cumulus cells; grade D are nude eggs> deformed oocytes and degenerative oocytes. The COCs of grade A and grade B have higher maturation rates.
5.The in vitro maturation rate of oocytes is obviously higher under 38.5℃ than under 37.0'C(p<0.05).
6.Medium 199 is the culture medium of in our experiments.. The following culture systems are all available and can make the in vitro maturation rate reach 78.9%, 84.2%: (l)M199+10%FBS+0.2mM Na-pyruvate+lmM antibiotic+10mg/ml GnRH +10mM Hepes +lmg/ml E2; (2)M199+20%FBS+0.2mM Na-pyruvate+lmM antibiotic+lOmg/ml GnRH.
7.The method of swim-up is effective for Wapiti(Cervus elaphus) sperm washing and capitation in vitro. The two centrifugalizing speeds in washing sperm being 2200rpm, 2000
rpm is best. The effects of operating in TALP sperm washing or fertilization medium(2) and in BO sperm washing or fertilization medium(2) are all better than in TALP sperm washing or fertilization medium(l) and in BO sperm washing or fertilization medium(l). The time of sperm living in vitro can reach to 24 hour when sperm cultured in BO fertilization medium(2), and the time in TALP-fertilization medium(2) is only 9h.
8.In vitro fertilization with BO or TALP can all make the ovum culturing to cleavage stage, and the fertilization rate are 66.7%, 70.6%. The effects of fertilizing in the two mediums are unobviously(p>0.05).
9.The early embryo of Wapiti(Cerv玸 elaphus) developed in embryo culture madium M199. The rate of the cells that have developed to 2-cell can reach to 50%.
引文
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54．俞颂东等．影响家畜超排效果因素和几种超排方法．草食家畜(季刊)，2002；29～32
55．袁运生等．精子获能机理及调控．黑龙江动物繁殖，2002；10(1)：15～17
56．曾申明等．卵泡直径和卵丘细胞对牛卵母细胞体外受精后发育潜力的影响．农业生物技术学报，2000；8(4)：357～360
57．张爱民编译．卵母细胞体外成熟体外受精生产犊牛．草食家畜，1995；9(3)：19～22
58．张涌等．哺乳动物卵泡卵母细胞的体外受精．河北农业大学学报，1996；19(3)：99～106
59．赵世臻等主编．中国养鹿大成．北京：中国农业出版社，1998
60．朱必才等．哺乳动物卵母细胞减数分裂研究进展．动物学杂志，2002；37(4)：79～83
61. Asher GW et al. Hybridization of Pere David's deer(Elaphurus davidianus)and red deer (Cervus elaphus) by artificial insemination. J Zool London, 1988;215: 197～203
62. Asher GW et al. Oestrous synchronization, semen collection and artificial insemination of farmed red deer(Cervus elaphus)and fallow deer(Dama dama). Anim Reprod Science, 1993;33: 241～265
63. Asher GW et al. Relationship between the onset of oestrous, the preovulatory surge in luteinizing hormone and ovulation following oestrous synchronisation and superovulation of farmed red deer(Cervus elaphus). J Reprod Fertil, 1992;96: 1～13
64. Bainbridge DRJ et al. Immunoneutralization with a monoclonal antibody attenuates the superstimulatory effects of PMSG on endocrine and ovarian responses in red deer(Cervus elaphus). Theriogenology, 1995;43: 1339～1350
65. Bainbridge DRJ et al. Role of oestrogen and prostaglandin F2 in premature luteal regression in monovulatory and superovalated red deer(Cervus elaphus). Biol Repred, 1996: 54: 347～354
66. Bainbridge DRJ et al. Successful in vitro fertilization of in vivo matured oocytes aspirated laparoscopically from red deer hinds (Cervus elaphus). Theriogenology, 1999;51(5): 891～898
67. Berg DK et al. Development of in vitro embryo production system for red deer(Cervus elaphus). Part1. Sperm motility and fertilisability when incubated on homologous epithelial oviductal monolayers. Anim. Reprod. Sci, 2001;submitted for publication
68. Berg DK et al. Development of in vitro embyo production systems for red deer(Cervus elaphus)part1. Effect of epithelial oviductal monolayers and heparin on in vitro sperm motility and penetration of in vitro matured oocytes. Anim Reprod Sci, 2002; 70(1-2): 65～
76.
69. Berg DK et al. Development of in vitro embryo production systems for red deer(Cervus elaphus). Part3. In vitro fertilisation using sheep serum as a capacitating agent and the subsequent birth of calves, hnim Reprod Sci, 2002;70(1-2): 85～98
70. Berg DK et al. Development of transvaginal oocyte pick-up for farmed red deer(Cervus elaphus). Theriogenology, 2000;53: 326 abstr
71. Berg DK et al. Pregnancies following the transfer of in vitro matured and fertilized red deer(Cervus elaphus)oocytes. Theriogenology, 1995;43: 166 abstr
72. Berg et al. Suceessful in vitro culture of early cleavage stage embryos recovered from superovulated red deer(Cervus elaphus). Theriogenology, 1995;44: 247～254
73. C. Caolan et al. Effeet of follicle size and quality on the ability of follicular to suppot cytoplasmic maturation of bovine oocytes. Mole Reprod Dev, 1996;43: 477～483
74. Cheng WTK et al. In vitro fertilization of pig and sheep ooeytes matured in vivo. Theriogenology, 1986;25: 146
75. Comizzoli P et al. Assessment of in vitro fertility of deer spermatozoa by heterotogous IVF with zona-free bovine oocytes. Theriogenology, 2001;56(2): 261～274
76. Comizzoli P et al. Successful in vitro production of embryos in the red deer(Cervus elaphus)and the sika deer(Cervus nippon). Theriogenology, 2001;55(2): 649～659
77. De Azambuja R M et al. Effect of gas atmosphere on in vitro maturation of bovine oocytes. Theiogenology, 1993;39: 184
78. Fukui Y et al. Effects of sera, hormones and granulose cells added to culture medium for in vitro maturation, fertilization, cleavage and development of bovine oocytes. J Reprod Fert, 1989;86: 501～506
79. Fukui Y et al. Effects of culture duration and time of gonadotropin addition on in vitro maturation and fertilization of red deer(Cervus elaphus)oocytes. Theriogenology, 1991;35: 499～512
80. Gandolfi F et al. Comparative analysis of calf and cow oocyte during in vitro maturation. Mol Reprod Dev, 1998;49(2): 168～175
81. Garde JJ et al. Postmortem assessment of sperm characteristics of the red deer during the breeding season. Arch Androl, 1998;41: 195～202
82. Garde JJ et al. Use of a triple-stain technique to detect viability and acrosome reaction in deer spermatozoa. Arch Androl, 1997;39: 1～9
83. Gordon I. Laboratory production of cattle embryos. Oxon: CAB International, 1994
84. Hunter AG et al. Stage-dependent effects of inhibiting riboncleic acids and protein synthesis on meiotic maturation of bovine oocytes in vitro. J Dairy Sci, 1987;70: 1646～ 1651
85. Jabbour HN et al. Effect of exogenous gonadotrophins on oestrus, the LH surge and the timing and rate of ovulation in ted deer(Cervus elaphus). J Reprod Fertil, 1994;100: 533～ 539
86. Jopson NB et al. Plasma progesterone concentrations in cycling and in ovariectomised red deer hinds: the effect of progesterone supplementation and adrenal stimulation. Anim Reprod Sci, 1990;23: 61～73
87. K. Goto et al. Oocyte maturation and fertilization. Ani Reprod Sci, 1992;28: 407～413
88. Krogenaes A et al. In vitro maturation and fertilization of oocytes from Norwegian semi-domestic reindeer(Rangifer tarandus). Theriogenology, 1994;41: 371～377
89. Kruip ThAM et al. Influence of temperation on the resumption meiosis of bovine oocytes in vitro. In: Rolland R. Hall EV Vam, Hillier SG, McNatty KP and Schoemaker J(eds), Follicular Maturation and Ovulation. International Congress Series, 1982: 282～288
90. Kruip ThAM et al. Potential use of Ovum Pick up for embryo production and breeding in cattle. Theriogenology, 1994;42: 675～684
91. Larsson B et al. In vitro oocyte maturation and fertilization in the bovine. Reprod Dom Anim, 1993;28: 161～163
92. Leibfried et al. Effects of fetal calf serum and bovine serum albumin on in vitro maturation and fertilization of bovine and hamster cumulus-oocytes complexes. Biol Reprod, 1986;35: 850～857
93. Lu KH et al. Pregnancy established in cattle by transfer of embryos derived from in vitro fertilization of follicular oocytes matured in vitro. Vet Rec, 1987;121: 259～260
94. Monfort SL et al. Successful intrauterine insemination of Eld's deer(Cervus eldi thamin) with frozen-thawed spermatozoa. J Reprod fertil, 1993;99: 459～465
95. Pieterse MC et al. Transvaginal ultrasound guided follicular aspiration of bovine oocytes. Theriogenology, 1991;35: 19～24
96. Pollard JW et al. In-vitro production of wapiti and red deer(Cervus elaphus)embryos. Theriogenology, 1995;43: 301
97. Revel F et al. Low developmental capacity of in vitro matured and fertilized oocytes from
calves compared with that of cows. J Reprod Fertil, 1995;103: 115～120
98. Sato E et al. Factors involving in vitro maturation of pig and cattle follicular oocytes cultured in vitro. Japan J Anim Reprod, 1977;23: 12～18
99. Simon L et al. Repeated bovine oocyte collection by means of a permanently rinsed ultrasound guided aspiration unit. Theriogenology, 1993;39: 312 abstr
100. Strzezek J et al. Seasonal changes in the chemical composition of red deer(Cervus elaphus) semen. Anim Reprod Sci, 1985;9: 195～204
101. Sun QY et al. Chronological events and ultrastructure of bovine in vitro fertilization. Theriogenology, 1994;41: 305
102. Wassarman PM et al. The mammalian ovum. In: Knobil E and Neill JD. (eds). The Physiology of Reproduction, Second Edition. New York: Raven Press, 1994: 79～122
103. Willard ST et al. Artificial insemination, hybridization and pregnancy detection in sika deer(Cervus nippon). Theriogenology, 1996;46: 779～789
104. Yang NS et al. In vitro fertilization and culture of bovine oocytes from stored ovaries. Therigenology, 1990;33: 352
105. Zomborszky Z et al. Sperm collection from shot red deer stags(Cervus elaphus)and the utilization of sperm frozen subsequently thawed. Acta Vet Hung, 1999;47: 263～270
2．廛洪武等．卵母细胞的不同对牛体外受精效果的影响．内蒙古大学学报(自然科学版)，1997；28(1)：112～116
3．陈大元主编．受精生物学．北京：科学出版社，2000
4．陈学进．牛卵母细胞体外成熟，体外受精和胚胎培养的研究进展．黑龙江畜牧兽医．1997；4：41～44
5．程世鹏等主编．特种经济动物常用数据手册．沈阳：辽宁科学技术出版社，2000
6．崔亚利等．牛卵母细胞体外成熟及体外受精的研究．河北农业大学学报，1999；1(1)：67～70
7．冯伯森等．成熟促进因子及其对卵母细胞成熟调控机制研究的新进展．动物学杂志，2003；38(1)：89～92
8．冯伯森等编著．动物细胞工程原理与实践．北京：科学出版社，2001
9．冯怀亮．哺乳动物胚胎工程[M]．吉林：吉林科学出版社，1994
10．高建明．牛体外受精卵体外培养研究进展．草食家畜，1996；3：14～19；4：21～23
11．郭志勤等编著．家畜胚胎工程．北京：中国科学技术出版社，1998
12．韩毅冰等．牛卵母细胞体外成熟的研究．生物技术，1995；5(5)：6～10
13．胡明信．活体采集卵母细胞技术．草食家畜(增刊)，2001；15～17
14．江金益．奶牛体外受精研究．江苏农业学报，1989；5(4)：1～8
15．李兵兵等．生长因子对卵母细胞体外成熟及其早期胚胎体外发育的影响．广西农业大学学报，1994；13(1)：11～15
16．李光鹏等．多肽生长因子与哺乳动物早期胚胎发育．黑龙江畜牧兽医，1994；(7)：41～43
17．李和平．中国茸鹿品种(品系)的生态种特征．东北林业大学学报，2001；29(5)：52～57
18．李丽立等主编．现代生物技术与畜牧业．北京：科学出版社，2002
19．李树静等．马鹿胚胎移植技术．中国兽医学报，2000；20(5)
20．刘红林等．哺乳动物卵母细胞的体外成熟．畜牧与兽医，1996；28(4)：180～182
21．刘建民等．牛卵泡卵体外成熟及其受精能力的研究．兽医大学学报，1989；9(4)：356～362
22．刘校光等．关于哺乳类动物精子获能的研究进展．阴山学刊，2000；15(4)：84～86，90
23．刘泽隆等．牛卵泡卵母细胞体外成熟的研究．西北农业大学学报，1999；27(2)：17～20
24．罗海玲等．血管内皮生长因子对牛卵母细胞体外成熟、体外受精、体外发育的影响．第十一届全国动物繁殖学术讨论会论文集，2002
25．马云等．影响牛卵母细胞体外成熟的因素．黄牛杂志，2000；2(1)：48～51
26．彭南妮等．哺乳动物卵泡卵母细胞的体外培养．湖南农业大学学报，1997；23(6)：594-598
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29．秦鹏春等编著．哺乳动物胚胎学．北京：科学出版社，2001
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31．石德顺等．第二信使因子对牛卵母细胞体外成熟的影响．中国兽医学报，1996；16(1)：80～85
32．石国庆．动物卵母细胞体外培养和体外受精技术研究进展．草食家畜，1998；12(4)：25～31
33．孙青原．牛卵母细胞体外成熟、冷冻及受精的研究．东北农业大学，1994
34．孙青原等．牛卵母细胞发育过程的细胞核变化．东北农学院学报，1990；21：234～238
35．孙青原等．体外受精的程序及超微结构研究．动物学报，1996；9(3)：303～308
36．孙青原等．性成熟前牛卵母细胞体外受精研究．中国畜牧杂志，1995；31(2)：21～22
37．孙少华等．哺乳动物体外受精研究进展及其在家畜育种中的应用．黄牛杂志，1998；4(24)：58～61
38．谭景和等．体外培养前黄牛卵母细胞的分类、形态结构及成熟能力的研究．中国农业科学，1989；22：83～88
39．谭世俭等．不同发情周期阶段和不同体积卵泡的卵母细胞对体外受精及其发育的影响．广西农学院学报，1991；10(2)：15～19
40．谭世俭等．受精温度对牛卵母细胞体外受精及随后胚胎发育的影响．广西农业大学学报，1994；13(1)：32～36
41．王锋．牛卵巢内卵母细胞采集、体外成熟与体外受精的研究．西北农业大学．陕西杨陵，1994
42．王海滨等．哺乳动物卵母细胞体外培养体系的研究进展．农业生物技术学报，2000；8(3)：297～310
43．王建辰等主编．动物生殖调控．安徽科学技术出版社，1998
44．王敏康等．卵母细胞、受精卵及早期胚胎质量判断方法的探讨．生殖医学杂志，2002；11(3)：171～ 175
45．文国艺．牛体外受精四种方法的比较．广西畜牧兽医，2000；16(2)：5～7
46．文国艺等．两种方法对牛体外受精及体外发育的影响．中国兽医学报，1996；16(5)：510～513
47．文国艺等．受精液中肝素和咖啡因的组合对牛卵母细胞体外受精的影响．广西农业大学学报，1994；13(1)：27～30
48．许厚强等．牛体外受精-屠宰牛卵泡卵的体外成熟培养．贵州农学院学报，1996；15(1)：26～30
49．旭日干等．屠宰母牛卵巢卵母细胞的体外受精和早期发生．内蒙古大学学报(自然版)，1989；20(3)：407～414
50．薛庆善主编．体外培养的原理与技术．科学出版社，2001
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52．杨庆章等．奶(黄)牛卵母细胞体外受精及体外发育的研究．生物技术，1991；1(3)：4～10
53．杨膺白等．卵丘细胞对牛卵母细胞体外成熟和体外受精的影响．广西农业大学学
报，1994；13(1)：16～20
54．俞颂东等．影响家畜超排效果因素和几种超排方法．草食家畜(季刊)，2002；29～32
55．袁运生等．精子获能机理及调控．黑龙江动物繁殖，2002；10(1)：15～17
56．曾申明等．卵泡直径和卵丘细胞对牛卵母细胞体外受精后发育潜力的影响．农业生物技术学报，2000；8(4)：357～360
57．张爱民编译．卵母细胞体外成熟体外受精生产犊牛．草食家畜，1995；9(3)：19～22
58．张涌等．哺乳动物卵泡卵母细胞的体外受精．河北农业大学学报，1996；19(3)：99～106
59．赵世臻等主编．中国养鹿大成．北京：中国农业出版社，1998
60．朱必才等．哺乳动物卵母细胞减数分裂研究进展．动物学杂志，2002；37(4)：79～83
61. Asher GW et al. Hybridization of Pere David's deer(Elaphurus davidianus)and red deer (Cervus elaphus) by artificial insemination. J Zool London, 1988;215: 197～203
62. Asher GW et al. Oestrous synchronization, semen collection and artificial insemination of farmed red deer(Cervus elaphus)and fallow deer(Dama dama). Anim Reprod Science, 1993;33: 241～265
63. Asher GW et al. Relationship between the onset of oestrous, the preovulatory surge in luteinizing hormone and ovulation following oestrous synchronisation and superovulation of farmed red deer(Cervus elaphus). J Reprod Fertil, 1992;96: 1～13
64. Bainbridge DRJ et al. Immunoneutralization with a monoclonal antibody attenuates the superstimulatory effects of PMSG on endocrine and ovarian responses in red deer(Cervus elaphus). Theriogenology, 1995;43: 1339～1350
65. Bainbridge DRJ et al. Role of oestrogen and prostaglandin F2 in premature luteal regression in monovulatory and superovalated red deer(Cervus elaphus). Biol Repred, 1996: 54: 347～354
66. Bainbridge DRJ et al. Successful in vitro fertilization of in vivo matured oocytes aspirated laparoscopically from red deer hinds (Cervus elaphus). Theriogenology, 1999;51(5): 891～898
67. Berg DK et al. Development of in vitro embryo production system for red deer(Cervus elaphus). Part1. Sperm motility and fertilisability when incubated on homologous epithelial oviductal monolayers. Anim. Reprod. Sci, 2001;submitted for publication
68. Berg DK et al. Development of in vitro embyo production systems for red deer(Cervus elaphus)part1. Effect of epithelial oviductal monolayers and heparin on in vitro sperm motility and penetration of in vitro matured oocytes. Anim Reprod Sci, 2002; 70(1-2): 65～
76.
69. Berg DK et al. Development of in vitro embryo production systems for red deer(Cervus elaphus). Part3. In vitro fertilisation using sheep serum as a capacitating agent and the subsequent birth of calves, hnim Reprod Sci, 2002;70(1-2): 85～98
70. Berg DK et al. Development of transvaginal oocyte pick-up for farmed red deer(Cervus elaphus). Theriogenology, 2000;53: 326 abstr
71. Berg DK et al. Pregnancies following the transfer of in vitro matured and fertilized red deer(Cervus elaphus)oocytes. Theriogenology, 1995;43: 166 abstr
72. Berg et al. Suceessful in vitro culture of early cleavage stage embryos recovered from superovulated red deer(Cervus elaphus). Theriogenology, 1995;44: 247～254
73. C. Caolan et al. Effeet of follicle size and quality on the ability of follicular to suppot cytoplasmic maturation of bovine oocytes. Mole Reprod Dev, 1996;43: 477～483
74. Cheng WTK et al. In vitro fertilization of pig and sheep ooeytes matured in vivo. Theriogenology, 1986;25: 146
75. Comizzoli P et al. Assessment of in vitro fertility of deer spermatozoa by heterotogous IVF with zona-free bovine oocytes. Theriogenology, 2001;56(2): 261～274
76. Comizzoli P et al. Successful in vitro production of embryos in the red deer(Cervus elaphus)and the sika deer(Cervus nippon). Theriogenology, 2001;55(2): 649～659
77. De Azambuja R M et al. Effect of gas atmosphere on in vitro maturation of bovine oocytes. Theiogenology, 1993;39: 184
78. Fukui Y et al. Effects of sera, hormones and granulose cells added to culture medium for in vitro maturation, fertilization, cleavage and development of bovine oocytes. J Reprod Fert, 1989;86: 501～506
79. Fukui Y et al. Effects of culture duration and time of gonadotropin addition on in vitro maturation and fertilization of red deer(Cervus elaphus)oocytes. Theriogenology, 1991;35: 499～512
80. Gandolfi F et al. Comparative analysis of calf and cow oocyte during in vitro maturation. Mol Reprod Dev, 1998;49(2): 168～175
81. Garde JJ et al. Postmortem assessment of sperm characteristics of the red deer during the breeding season. Arch Androl, 1998;41: 195～202
82. Garde JJ et al. Use of a triple-stain technique to detect viability and acrosome reaction in deer spermatozoa. Arch Androl, 1997;39: 1～9
83. Gordon I. Laboratory production of cattle embryos. Oxon: CAB International, 1994
84. Hunter AG et al. Stage-dependent effects of inhibiting riboncleic acids and protein synthesis on meiotic maturation of bovine oocytes in vitro. J Dairy Sci, 1987;70: 1646～ 1651
85. Jabbour HN et al. Effect of exogenous gonadotrophins on oestrus, the LH surge and the timing and rate of ovulation in ted deer(Cervus elaphus). J Reprod Fertil, 1994;100: 533～ 539
86. Jopson NB et al. Plasma progesterone concentrations in cycling and in ovariectomised red deer hinds: the effect of progesterone supplementation and adrenal stimulation. Anim Reprod Sci, 1990;23: 61～73
87. K. Goto et al. Oocyte maturation and fertilization. Ani Reprod Sci, 1992;28: 407～413
88. Krogenaes A et al. In vitro maturation and fertilization of oocytes from Norwegian semi-domestic reindeer(Rangifer tarandus). Theriogenology, 1994;41: 371～377
89. Kruip ThAM et al. Influence of temperation on the resumption meiosis of bovine oocytes in vitro. In: Rolland R. Hall EV Vam, Hillier SG, McNatty KP and Schoemaker J(eds), Follicular Maturation and Ovulation. International Congress Series, 1982: 282～288
90. Kruip ThAM et al. Potential use of Ovum Pick up for embryo production and breeding in cattle. Theriogenology, 1994;42: 675～684
91. Larsson B et al. In vitro oocyte maturation and fertilization in the bovine. Reprod Dom Anim, 1993;28: 161～163
92. Leibfried et al. Effects of fetal calf serum and bovine serum albumin on in vitro maturation and fertilization of bovine and hamster cumulus-oocytes complexes. Biol Reprod, 1986;35: 850～857
93. Lu KH et al. Pregnancy established in cattle by transfer of embryos derived from in vitro fertilization of follicular oocytes matured in vitro. Vet Rec, 1987;121: 259～260
94. Monfort SL et al. Successful intrauterine insemination of Eld's deer(Cervus eldi thamin) with frozen-thawed spermatozoa. J Reprod fertil, 1993;99: 459～465
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