Effect of culture stages supplemented with different antioxidants on the development of parthenogenetic and ICSI porcine embryos

• Main Authors: Chen, Tse-Yu, 陳則佑
• Other Authors: Shen, Perng-Chih
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/ss36eb

碩士 === 國立屏東科技大學 === 動物科學與畜產系所 === 106 === In vitro production (IVP) of mammalian embryos are inclusive of in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC).At present, the ambient oxygen concentration used during the in vitro production of this embryos are about 20%, which is already a high oxygen environment comparing with the oxygen concentration of about 5 to 8% in vivo, and reduce the development capability of the embryo. In addition, during intracytoplasmic sperm injection (ICSI), if the sperm injection is too close to the oocytes nucleus, the development of ICSI embryos will be reduced. However, the effects of antioxidant types and culture stages to the subsequent development of oocytes and embryos in porcine, as well as sperm injection location on the development of embryos in ICSI porcine have not yet been discussed in detail. Based on this aspect, porcine oocytes were used as experimental materials in this study. Firstly, different concentration of glutathione (GSH) and β-mercaptoethanol (β-ME) are studied in in vitro maturation and in vitro culture on the developmental ability of porcine oocytes after parthenogenetic activation. In study 2, the in vitro culture conditions of porcine oocytes and ICSI porcine embryos were explored with the specific concentration of GSH or β-ME obtained from study 1 to further evaluate the effects of both types and sperm injection position for the in vitro development ability of ICSI porcine embryos. The results of IVM stage showed that IVM medium with 0-5 mM GSH group porcine oocytes shared similar blastocyst rates (15.8-25.0%) after parthenogenetic activation. IVM medium with 10 μM β-ME, the blastocyst rate (35.9%) was significantly higher (P < 0.05) than control group (20.5%) and which was not supplemented with β-ME. However, the blastocyst rates of IVM medium with 25 μM (23.4%), 50 μM β-ME and control group (23.5%) had no significant differences (P > 0.05). IVM medium with 1 mM GSH and 10 μM β-ME of porcine oocytes which are activated sharing similar blastocyst rates (24.4% vs. 18.7). The results at the IVC stage showed that in the IVM stage oocytes were cultured at 10 μM β-ME condition, the blastocyst rate of IVC culture medium with 0.5 mM GSH (41.7%) after parthenogenetic activated porcine embryos showing no significant difference (P > 0.05) from control group (32.4%), but significantly (P < 0.05) higher than IVC medium with 1 mM (27.1%) and 5 mM (26.6%) GSH treatment group. However, the blastocyst rate of IVC medium with 10 to 50 μM β-ME (12.1-20.3%) and control group (22.4%) after parthenogenetic activation of porcine embryo shared no significant difference (P > 0.05), and IVC medium with 0.5 mM GSH and 10 μM β-ME after parthenogenetic activation of porcine embryo also possess similar blastocyst rate (21.1% vs. 15.6). The results of Study 2 show that oocytes culture in the IVM medium with 10 μM β-ME NCSU-23, NCSU-23-hCG, and TCM-199-hCG medium, The blastocyst development rate of parthenogenetic embryos (14.1-19.4%) had no significantly different (P > 0.05) among the above 3 treatment groups, but in TCM-199-hCG group shared the highest blastocyst rate (19.4%). After comparison of sperm injection position and semen let stand upper and lower sperm as a source of ICSI sperm injection on embryo development capability. According to the results, the blastocyst rate of ICSI porcine embryos produced by injecting sperm near the first polar body of oocytes after activation was significantly lower than(P < 0.05) that sperm injection position away from the first polar body of oocytes (3.1 vs. 15.0%). Whereas, under the condition of sperm injection position away from the first polar body of oocytes and selection of the sperm activity, no matter what sort of choice is located in the upper (13.8%) or lower (12.4%) of the sperm, porcine production by ICSI embryos have similar (P > 0.05) blastocyst rate after activation. Taken together, both the IVM stage of porcine oocytes and the IVC stage of embryo culture may be supplemented with 10 μM β-ME to the culture medium to increase the in vitro blastocyst rate of parthenogenetic activation porcine embryos; However, in the production of ICSI porcine embryos, the sperm injection position should be away from the first polar body of the oocyte, but if active sperm is selected, the sperm in the upper and lower layers after standing can be implemented.