Bondioli K R, Wright R W
J Anim Sci. 1983 Oct;57(4):1006-12. doi: 10.2527/jas1983.5741006x.
In vitro fertilization of ovine ovulated and ovarian oocytes was attempted after capacitation of ejaculated spermatozoa by a variety of methods. All sperm incubations and oocyte cultures were performed in a modified Krebs Ringer bicarbonate (KRB) medium. High ionic strength (HIS) medium was prepared by adding NaCl to provide an osmolarity of approximately 370 mOsmol/kg. All oocytes were obtained from progestogen-synchronized and follicle-stimulating hormone-treated ewes. Ovulated oocytes were recovered 64 h after progestogen withdrawal. Ovarian oocytes were recovered 45 h after progestogen withdrawal. Oocytes were incubated with spermatozoa in microdrops for 6 h, transferred to fresh medium and cultured for 48 h. In the first series of experiments, capacitation was attempted by either preincubation in KRB, washing spermatozoa with KRB medium or treatment of spermatozoa with HIS medium. Spermatozoa washed with KRB penetrated three of 61 (5%) oocytes and spermatozoa incubated in HIS medium penetrated eight of 209 (4%) oocytes. In the second series of experiments, spermatozoa were capacitated by incubation with ovine cumulus cells or in the uterus of a rabbit. A 33% fertilization rate was observed with spermatozoa recovered from the rabbit uterus and a 28% rate with spermatozoa incubated with cumulus cells. Treatment with HIS medium previously shown to capacitate bovine and rabbit spermatozoa failed to capacitate ram spermatozoa; however, fertilization was observed after capacitation by cumulus cells or in the rabbit uterus.
通过多种方法使射出的精子获能后，尝试对绵羊排卵的卵母细胞和卵巢卵母细胞进行体外受精。所有精子孵育和卵母细胞培养均在改良的 Krebs 林格碳酸氢盐（KRB）培养基中进行。高离子强度（HIS）培养基通过添加氯化钠制备，使其渗透压约为 370 mOsmol/kg。所有卵母细胞均取自经孕激素同步化处理和促卵泡激素处理的母羊。在撤去孕激素 64 小时后收集排卵的卵母细胞。在撤去孕激素 45 小时后收集卵巢卵母细胞。将卵母细胞与精子在微滴中孵育 6 小时，转移至新鲜培养基中并培养 48 小时。在第一系列实验中，尝试通过在 KRB 中预孵育、用 KRB 培养基洗涤精子或用 HIS 培养基处理精子来使其获能。用 KRB 洗涤的精子穿透了 61 个卵母细胞中的 3 个（5%），在 HIS 培养基中孵育的精子穿透了 209 个卵母细胞中的 8 个（4%）。在第二系列实验中，精子通过与绵羊卵丘细胞共同孵育或在兔子子宫中孵育来获能。从兔子子宫中回收的精子受精率为 33%，与卵丘细胞共同孵育的精子受精率为 28%。先前已证明能使牛和兔子精子获能的 HIS 培养基处理未能使公羊精子获能；然而，在通过卵丘细胞或在兔子子宫中获能后观察到了受精现象。
