Fertilization and subsequent development in vitro of pig oocytes inseminated in a modified tris-buffered medium with frozen-thawed ejaculated spermatozoa
Fertilization and subsequent development in vitro of pig oocytes inseminated in a modified tris-buffered medium with frozen-thawed ejaculated spermatozoa
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DOI：
10.1095/biolreprod57.4.729
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发表时间：
1997-10-01
影响因子：
3.6
通讯作者：
Day, BN
中科院分区：
文献类型：
--
作者：
Abeydeera, LR;Day, BN
本研究探讨了猪卵母细胞的穿透性冻融射出的精子，制备的颗粒法，共孵育在一个修改的Tris缓冲液。还测定了受精卵母细胞的后续胚胎发育。将猪卵母细胞-卵丘复合体在无BSA的北卡罗来纳州州立大学（NCSU）23培养基中培养20-22小时，所述培养基含有猪卵泡液（10%）、半胱氨酸（0.1 mg/ml）和激素补充剂（eCG和hCG：各10 IU/ml）。然后将它们在相同的培养基中培养，但不添加激素补充剂，再培养20-22小时。培养后，去除卵丘细胞，将卵母细胞与三种不同浓度（1 × 10（5）、5 × 10（5）和1 × 10（6）/ml）的精子共孵育12小时（实验1）。在实验2中，卵母细胞与精子（5 × 10（5）/ml）共孵育3、6、9和12 h。在实验3中，在与5 × 10（5）/ml浓度的精子共孵育6 h后，将卵母细胞转移到NCSU 23 + 0.4%BSA培养基中。在48和144小时，分别评价卵裂率和囊胚形成率。用1 × 10（5）/ml的精子授精，卵母细胞的精子穿透率为40%，多精受精率为16%。每个卵母细胞的平均精子数（MNS）为1.2 +/- 0.1。在5 × 10（5）和1 × 10（6）/ml时，穿透率（84-87%）和多精受精（57-64%）增加（p < 0.001），两种浓度之间无差异。但随着精子浓度的增加，每卵母细胞的MNS增加（p < 0.05）。3 h时渗透率为31%，6-12 h时增加（p < 0.001）（80-88%），这些时间点之间无差异。多精受精以时间依赖性方式增加（p < 0.05），直至9 h，9 h和12 h之间无差异。与3 h相比，9 h和12 h的MNS/卵母细胞增加（p < 0.05），6 h无显著差异。培养48 h后，卵裂率为40%，144 h囊胚率为19%。本研究介绍了精液冷冻保存的精液丸法和成功的体外受精的猪卵母细胞的冷冻解冻精子，随后发育到囊胚期。
The present study examined the penetrability of pig oocytes by frozen-thawed ejaculated boar spermatozoa, prepared by the pellet method, coincubated in a modified Tris-buffered medium. Subsequent embryonic development of fertilized oocytes was also determined. Porcine oocyte-cumulus complexes were cultured in BSA-free North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml), and hormonal supplements (eCG and hCG: 10 IU/ml each) for 20-22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20-22 h. After culture, cumulus cells were removed and oocytes were coincubated for 12 h with three different (1 x 10(5), 5 x 10(5), and 1 x 10(6)/ml) sperm concentrations (experiment 1). In experiment 2, oocytes were coincubated with sperm (5 x 10(5)/ml) for 3, 6, 9, and 12 h. In experiment 3, at 6 h after coincubation with sperm at 5 x 10(5)/ml concentration, oocytes were transferred into NCSU 23 + 0.4% BSA medium. At 48 and 144 h, cleavage and blastocyst formation rates, respectively, were evaluated. Insemination with 1 x 10(5)/ml resulted in a 40% sperm penetration rate of oocytes with 16% polyspermy. Mean number of sperm (MNS) per oocyte was 1.2 +/- 0.1. At 5 x 10(5) and 1 x 10(6)/ml, penetration rate (84-87%) and polyspermy (57-64%) increased (p < 0.001), with no difference between the two concentrations. However, MNS per oocyte increased (p < 0.05) with increasing sperm concentration. Penetration rate was 31% at 3 h and increased (p < 0.001) at 6-12 h (80-88%), with no difference between these time points. Polyspermy increased (p < 0.05) in a time-dependent manner up to 9 h, with no difference between 9 and 12 h. Compared to 3 h, MNS per oocyte increased (p < 0.05) at 9 and 12 h, with no mean difference at 6 h. At 48 after culture, the cleavage rate was 40%, and at 144 h, the blastocyst rate was 19%. This study describes the cryopreservation of ejaculated boar semen by the pellet method and the successful in vitro fertilization of pig oocytes by frozen-thawed spermatozoa with subsequent development to the blastocyst stage.