| Porcine oocyte maturation and fertilization in vitro were studied in this paper, in order to obtain high quality of mature oocyte, reduce polyspermy and improve the quantity of blastocyst. So as to establish a relatively perfect system of embryo production technology in vitro.1. Study on the effects of gonadotropin on porcine oocyte maturation and Parthenogenetic embryo development in vitro: The present study was designed to explore the influencing of hornone(FSH, LH) and action time in porcine oocytes maturation medium on oocytes maturation and Parthenogenetic embryo development in vitro. In the experiment, the control is that the concentration of FSH, LH were 10ug/m L in medium and the action time is 42 h. The best group was determined by appreciating quality indexes of oocyte maturation, such as process of nuclear, survival rates, polar body rates, GSH contents in single oocyte and so on, after oocyte being cultured for 42 h in vitro. Then we further investigated oocytes cleavage and blastocyst rates after parthenogenetic activation to assure that the best concentration of hornone and the action time had benefits for embryo development. The results showed that the nuclear of four groups are all mature, there were no significant differences in survival and polar body rates among the four groups(P>0.05). However, GSH contents in single oocyte of treatment groups were significantly higher than the control(P<0.05). Furthermore, Oocyte cleavage and blastocyst rates in 1ug/m L hornone and 42 h treated group was greatly improved when compared to the control after parthenogenetic activation(P<0.05). The results demonstrated that 1ug/m L hornone concentration supplemented in porcine oocyte whole maturation medium not only could improve maturation qualities of oocyte, more importantly, also could enhance developmental potential of early embryos in vitro.2. Study on the effects of melatonin on porcine oocyte maturation and Parthenogenetic embryo development in vitro: The present study was designed to explore the influencing of melatonin in porcine oocytes maturation medium on oocytes maturation and Parthenogenetic embryo development in vitro. The best melatonin(MT) concentration supplemented during porcine oocytes maturation was determined by appreciating quality indexes of oocyte maturation, such as survival rates, polar body rates, GSH contents in single oocyte, mitochondria and lipid droplet distribution of oocytes and so on, after oocyte being cultured for 42 h in vitro. Then we further investigated oocytes cleavage and blastocyst rates after parthenogenetic activation to assure that the best MT concentration had benefits for embryo development. The results showed that there were no significant differences in survival and polar body rates among the four groups(P>0.05); However, GSH contents in single oocyte of melatonin groups were significantly higher than the control(P<0.05). Furthermore, the rates of oocyte with better maturation distribution of mitochondria and lipid droplets were both significantly higher in melatonin groups than those in the control(P<0.05). Oocyte cleavage and blastocyst rates in 10-6mol/L and 10-9mol/L melatonin groups were greatly improved when compared to the control after parthenogenetic activation(P<0.05). The results demonstrated that 10-9 mol/L ~10-6 mol/L melatonin concentrations supplemented in porcine oocyte maturation medium not only could improve maturation qualities of oocyte, more importantly, also could enhance developmental potential of early embryos in vitro.3. Study of genes expression in MPF and MAPK of oocytes on melatonin: The experiment adopts two methods to extract RNA, and m RNA expression was investigated for the MPF and MAPK gene in porcine oocytes. Experiments were divided into four different melatonin concentrations. The results were showed as followed: m RNA expressions in MPF and MAPK of 10-9 mol/L melatonin concentration supplemented in porcine oocyte maturation medium were greatly higher than the control(P<0.05).4. Study on the effects of caffeine on porcine sperm capacitation and fertilization in vitro: The present study was designed to explore the influencing of caffeine in vitality, survival and fertilization of porcine sperm. In the experiments, the control is 0mmol/L caffeine concentration, porcine sperm is capacited in different concentrations(0, 2.5, 5, 10, 20 mmol/L). The vitality was tested every 30 min until lower than 0.6 and the strongest vitality was utilized fertilization. The results were showed as followed: 1) The vigor is less than 0.6 when the time of sperm capacitation is 90 min in 10 and 20 mmol/L caffeine concentration, however the others is higher than 0.6. 2) The rate of sperm penetrated into the egg was improved during caffeine, however, the rate of polyspermy of 20 mmol/L caffeine concentration is significant higher than the control(P<0.05). 3) 2.5~5.0 mmol/L caffeine concentrations supplemented in insemination medium could enhance developmental potential of early embryos in vitro.The quality of oocyte maturation was improved by culture system establishment of IVM. All of these had important reference value on porcine embryonic biotechnology. |
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