Establishment Of Donor Somatic Cell Lines And Oocyte In Vitro Maturation And Parthenogenetic Activation Of Putian Black Pig

Putian black pig had characterized by good quality, strong adaptability, high litter size, strong disease resistance as well as feeding with coarse fodder. As endangered species, Putian black pig urgent to been protection.Developing with somatic cell nuclear transfer technique, its important application value and foreground in protecting species. this research focused on main items of Pig somatic nuclear transfer systematically such as somatic donors,recipient oocyte.1. Establishment of donor somatic cell linesAdult ear skin fibroblast cell lines were set up by using explant-seeding method and enzyme digestion method. Pig oviduct epithelium cell lines were also established by scraping and trypsin digestion method and cumulus cells from follicular fluid. This experiment studied and compared advantages and shortcomings of the preparation methods. Due to protecting species ,we choose the Adult ear skin fibroblast cell lines were set up by using explant-seeding method.2. oocyte in vitro maturation and parthenogenetic activationThe system of oocytes maturation in vitro (IVM) had been optimized.(1) the rate of oocytes maturation was significant higher after cultured in NCSU-23(67.83%) than in TCM-199(58.14%)(P<0.05); (2) For oocytes IVM , NCSU-23+10% FBS+10%pFF(77.21%)seemed a little superior then NCSU-23+10%pFF(72.02%). The rate of blastocyst of them(30.38% and 29.70%) had significant differences(P<0.05) with NCSU-23+10%FBS(21.32%).(3) NCSU-23 medium with or without epidermal growth factor (EGF) had no much differences, but the effect with EGF was better after parthenogenetic activation.(4)The rate of oocytes maturation , rate of cleavage and blastocyst after parthenogenetic activation had no much differences when NCSU-23 medium storage at 5℃and -18℃Repeated parthenogenetic activation experiments confirmed that in the different activation conditions, effects of both NCSU-23 and TCM-199 were similar on parthenogenetic activation (P>0.05),and 2.0kv/cm,60μs,1DC was suitable to improve cleavage rate and blastocyst rate.