| 1. The aspiration method was used for recovering the cumulus-complexes (COCs) from surface-visible follicles of porcine ovaries from slaughterhouse. Those COCs that have compact cumulus cells and homogeneous oocyte plasma (A and B grade) were chosen for in vitro maturation (I VM). The effect of the culture medium composition, culture methods, follicle size, and transportation temperature on the IVM were studied. The results were as followings: (1) Four groups of culture medium with different hormone supplement: group A (M199+10%FBS+10IU/ml PMSG+10IU/ml hCG+2.5IU/ml FSH), B (M199+ 10%FCS+10IU/ml PMSG+10IU/ml hCG+10%PFF), C (M199+10%FCS+10IU/ml hCG ), and D (M199+10IU/ml PMSC+25 mg/ml BSA ) were used in our experiments, the IVM rate of porcine oocyte was 61.8%, 64.0%, 38.3%, and 66.1%, respectively. It showed significant difference between group C and other three groups (p<0.01). The IVM porcine oocytes of group D was observed with smaller perivitelline space and first polar body than other groups. (2) Different basic medium (Ml99, mM199) had significant difference (p<0.01) in the rate of IVM, which was 60.3%, 79.3% respectively. (3)With or without serum in the maturation medium, the porcine oocyte IVM rate was 33.8% and 60.3%, respectively, with significant difference (p<0.01). The cumulus expansion of IVM oocytes from group without serum was insufficient; (4)Three groups was divided in the light of the diameter of the porcine oocytes: group A <2 mm, B from 2 to 8 mm, C > 8 mm. The IVM rate of those groups was 21.7%, 64.9%, 82.4% respectively, which showed significant difference (p < 0.01) between group A and other two groups. There was more fragments in the perivitelline space of oocytes of group C than other groups, it showed that the oocyte quality of group C was very poor. (5) Different ovaries transportation temperature (25-30℃, 30-35℃, 35-39℃), obtained different rate of the porcine oocyte IVM in our experiments. It showed significant difference among them, and suggested a linar relationship between transportation temperature and the porcine oocyte IVM rate. Particularly, when the transportation temperature was 35-39℃, manipulation temperature was 37℃, the IVM rate reached to 60.3%. The lower ovary's transplantation temperature was, the poorer quality of oocytes was.2. The effect of electric field strength pulse number and the IVM oocyte age on the porcine oocyte parthenogenetic activation was studied. When the electric field strength increased, the oocyte lyse rate and the parthenogenetic embryos cleavage rate increased gradually, when the direct current field strength was 150v/mm (duration 60us), the result was better (no lysed oocyte, embryo cleavage rate 81.6%); when the field strength was 150v/mm (duration 60us), multiple pulse (2 or 3) did not increase the parthenogenetic embryo cleavage rate significantly. But when pulse number increased, the number of lysed oocyte increased. When two electric pulses was used better result was obtained (no lysed oocytes, cleavage rate 71.4%). When the oocytes after IVM for 40,44,48 and 52h, activated with 2 pulses of direct current field strength (150v/mm;dutation 60us, interval 3s),the parthenogenetic embryo cleavage rate was 66.7%,75.0%,82.8%and 84.0% respectively, and the oocyte lyse rate 0.0%,0.0%,0.0% and 10.7%. Better result was obtained from oocytes after IVM for 48h,.3. The NCSU-23 was a better medium for culturing the porcine parthenogenetic embryo in vitro, than M199+10% FBS, which did not depend on cumulus cell feeder layer. The clearage rate of the parthenogenetic embryo cultured in NCSU-23 was 93.2%, blastocyt rate 31.8% which was significantly higher than M199+10%FBS.4. Human embryonic fibroblasts (HEF) and adult skin fibroblasts (hASF) were isolated by trypsinization and explant culture respectively. These two cell lines were purified according to the different attachment time and sensitivity to TE between fibroblasts and epithelial cells. These cells were cryopreserved in liquid ni...
|
PDF Full Text Request