Studies on in vitro fertilization and embryo production by intracytoplasimc sperm injection (ICSI) in cattle

本研究旨在通过研究牛精子和卵母细胞的各种条件，建立牛胞浆内单精子注射（ICSI）技术。对于精子，(1)精子预处理，(2)精子固定化，(3)聚乙烯醇吡啶酮（PVP）浓度，对于卵母细胞，(1)卵母细胞成熟期，(2)离心使卵浆脂质极化，(3)卵母细胞活化方法，进行了研究，以证明牛在ICSI后不需要卵母细胞活化的假设，并将囊胚转移到受体母牛身上以获得正常的小牛。所得结果为：(1)公牛差异改变了常规体外受精（IVF）和ICSI的结果。也就是说，用低IVF率的公牛精液进行ICSI可以提高雄性原核形成率。(2)不同精子类型（活动精子、静止精子和死亡精子）的雄性原核形成率无显著差异。钙离子载体和精子固定化均能提高受精率。(3)离心、4% PVP浓度和剪去精尾可提高卵母细胞存活率和正常受精率。随后的卵裂率为72%，囊胚发育率为23%。(4)在未激活卵母细胞的情况下，将8个ICSI囊胚移植后，7头母牛中有4头怀孕，产下5头正常犊牛。这些结果表明，建立了一种牛ICSI方法，并表明在牛ICSI后不需要卵母细胞活化。

The research was aimed to establish an intracytoplasmic sperm injection (ICSI) in cattle by studying various conditions for spermatozoa and oocytes. For spermatozoa, (1) sperm pre-treatment, (2) sperm immobilization (3) concentrations of polyvinylpyrolidone (PVP), and for oocytes, (1) oocyte maturation period, (2) centrifugation to polarize the ooplasmic lipid, (3) oocyte activation methods, were investigated to prove a hypothesis that oocyte activation after ICSI is not necessary in cattle, and the blastocysts were transferred to recipient heifers to obtain normal calves. The results obtained were; (1) Bull difference varied the outcomes by conventional in vitro fertilization (IVF) and by ICSI. Namely, male pronuclear formation rate was improved by ICSI using bull semen with low IVF rates. (2) There was no significant difference in the male pronuclear formation rate among the sperm types (motile, immotile or dead sperm). Ca ionophore and sperm immobilization enhanced the fertilizability. (3) Oocyte survival and normal fertilization rate were improved by centrifugation, 4% PVP concentration and sperm-tail cutting. The subsequent cleavage and blastocyst development were 72% and 23%, respectively. (4) After transfer 8 blastocysts derived by ICSI without oocyte activation, 4 out of 7 heifers were pregnant and gave 5 normal calves. These results indicate that a bovine ICSI method was been established, and showed that oocyte activation after ICSI is not necessary in cattle.