牛体外受精胚不同发育阶段外源性氨基酸消耗量的研究

The freezing viability and pregnancy rate of bovine IVF embryo are lower than that of embryo derived from in vivo. It's due to the culture condition for embryo development resulted from the limited understanding about the development mechanism of early embryo. Amino acids have been shown to improve the development of bovine IVF embryos. But amino acid stock solutions of commercial product were designed originally for cell culture. Firstly, this study was conducted to measure the levels of free amino acids in bovine oviductal fluid and uterine fluid at different preimplantation stages. Secondly, amino acids at physiological concentrations were supplemented into chemically defined medium SOF and bovine IVF embryo developmental potential was investigated. Consumption of amino acids by bovine IVF embryo at different development stages were measured, and effects of that some kinds of amino acid were absent in culture medium on consumption of the other amino acids and the freezing viability of embryo were investigated. The results:(1)The concentration of free amino acids in estrous day3 oviductal fluid(OF)(31.6pmol/L) was higher than that in medium supplemented commercial amino acid stock solution(MC)(3.4pmol/L), and βAla,Tau,Orn and Cit that were not included in commercial stock solution were identified in OF. The content of non-essential and essential amino acids were 27.8 and 2.7 pmol/L in OF, and 0.7 and 2.7 pmol/L in MC. (2) Amino acids at physiological concentration improved embryo development (blastocyst development rates 20-30%, hatched blastocyst rates 10-20%, re-expanded rates of blasocyst after freezing and thawing 75-90%) compared with commercial stock solution (10-20%, 0-5%, 60-70%). (3) During 1_8-cell stage, embryo released 23 kinds of amino acids, and the total releasing was 6372 pmol/embryo. During 8-cell_morula stage, embryo continued to release 21 kinds of amino acids. Meanwhile embryo began to absorb Gln and Arg. The trend of this stage was release, and the total releasing was 2407 pmol/embryo. After morula stage, embryo began to absorb 15 kinds of amino acids including Glu, Gly, Gln, Arg and so on, and release 8 kinds of amino acids. The mainstream of amino acid metabolism during this stage was uptake, and the total uptake was 1124 pmol/embryo. This results indicated that bovine oocyte have rich endogenous free amino acids reserves. (4) Consumptions of the other amino acids were affected by the absence of Gly or Gln, but the blastocyst development rates, hatched blastocyst rates and re-expanded rates of blastocyst after freezing and thawing weren't affected. Neither blastocyst rates，hatched blastocyst rates and re-expanded rates of blastocyst nor consumptions of the other amino acids were affected by the absence of Glu. The results indicated that the embryo exhibit a degree of plasticity regarding the use of amino acids, it could compensate the lack of particular amino acid in the medium by endogenous amino acids reserves or increasing the uptake of another. This research was helpful to elucidate the mechanism of bovine early embryo development, and improve the culture system and viability of bovine IVF embryo

拟采用高效液相色谱仪检测牛体外受精胚胎在不同发育阶段对２１种氨基酸的消耗量，阐明胚胎发育过程中利用外源氨基酸的规律，确定与胚胎质量及抗冻性密切相关的氨基酸种类及剂量。本项目研究对揭示早期胚胎的发育机制，进一步改善体外培养环境，提高牛体外受精胚胎的质量及低温耐受性均具有重要的理论及实际意义。