| Dairy embryos used for embryo transfer (ET) can be flushed from donors in vivo. But up to date, in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) are becoming the alternative methods of production bovine embryos for ET. This study was composed of four experiments. Experiment 1 was carried out to investigate the factors which affected the efficiency of bovine oocytes in vitro fertilization with sex-sorted sperm; Experiment 2 was carried out to investigate the effects of activation method, synchronization of donor somatic cells and culture medium on the development of bovine SCNT embryos; Experiment 3 was carried out to investigate the effects of controlled freezing method (traditional) and open-pulled straw (OPS) vitrification method on the ATP content and reactive oxygen species (ROS) level of bovine IVF and SCNT blastocysts; and Experiment 4 was carried out to detect the expression level of CYBT gene encoded by the mitochondria in equine-bovine iSCNT embryos produced by HMC and microinjection with the quantitative RT-PCR.The results were as followings:Experiment 1The present experiment was designed to investigate main factors that influenced the result of IVF with bovine sex-sorted sperm. The number of cumulus cells(intact, partial removal, complete removal), semen from different bulls (A, B and C), volume of IVF drops (30μl-100μl) and sperm concentration (0.3×10~6/mL -1×10~6/mL) were analyzed. The results showed that :(1) the cleavage rate of oocytes with partial removal of cumulus cells after fertilization in vitro (67±4.74%) was significantly higher than that of oocytes with intact cumulus cells (41±2.23%) or complete removal of cumulus cells (37.50±2.88%) (P<0.05); (2) the blastocyst rate (26.53±3.31%)of oocytes fertilized with sex-sorted sperm of bull B was significantly higher than those of bull A (17.65±3.65%)and C (16.67±2.36%)(P<0.05); (3) the blastocyst rate of oocytes fertilized in 100μl (23.38±4.47%), 80μl (21.05±3.70%),60μl (20.41±4.33%) and 50μl (21.67±3.92%) drops (P>0.05) showed no significant difference, but was significantly higher than those fertilized in 40μl (13.79±2.02%) or 30μl (12.12±3.73%) (P<0.05); (4) the blastocyst rate of oocytes fertilized with the sperm number of 1×10~6/mL (27.27±3.49%), 0.8×10~6/mL (24.68±2.34%),0.5×10~6/mL (22.58±2.17%) groups (P>0.05) showed no significant difference, but were significantly higher than those fertilized with 0.3×10~6/mL sperms (16.13±3.46%) group (P<0.05).Experiment 2In this experiment, the effects of activation method, synchronization of donor somatic cells and culture medium on the development of bovine SCNT embryos were investigated. The results showed that: (1) twenty cell lines of dairy top-bulls and two cell lines of high-milk-yield Holstein cows were established, respectively. (2) the rate of cleavage and blastocyst of reconstructed embryos activated by A23187+6-DMAP and A23187+CHX had no significant difference. However the quality of blastocysts developed from cloned embryos activated by A23187+6-DMAP was better than that by A23187+CHX. (3) the cleavage rate (83.65%) and blastocyst rate (34.36% ) of reconstructed embryos cultured in mCR1aa medium were significantly higher than that cultured in CR1aa and SOF, respectively. (4) there was no significant difference between the development rates of the reconstructed embryos cloned from donor cells of serum starvation and that of embryos cloned from contact-inhibited donor cells. (5) the pregnant rate of Day-7 bull cloned embryo was 37.0% checked by rectal palpation at 60 days after ET when two embryos were transferred to one recipient. Meanwhile, the pregnant rate of Holstein heifers recipients was higher than that of Simmental cows as recipients.Experiment 3This experiment was designed to investigate the influence of cryopreservation on ATP content and ROS level of bovine blastocysts produced by IVF and SCNT. The results showed that: (1) the survival rates of IVF and SCNT blastocysts culture in vitro after frozen-thawed by OPS vitrification method (92.24±4.54%, 78.71±5.91%) were significant higher than that of controlled freezing method (81.56±2.33% and 47.89±5.83%) (P<0.05), respectively. (2)The ATP contents of IVF blastocysts (0.62±0.04 pmol) and SCNT blastocysts (0.30±0.01 pmol) frozen-thawed by OPS vitrification method were significantly higher than that of controlled freezing method(0.43±0.06 pmol, 0.21±0.02 pmol), but were significantly lower than that of fresh IVF and SCNT (control ) (0.74±0.05 pmol and 0.39±0.01 pmol ) (P<0.05), respectively. (3) The ROS level of fresh IVF blastocysts (47.33±3.56 cps) and SCNT blastocysts (26.44±1.49 cps) was significantly lower than that of blastocysts frozned-thawed by OPS vitrification method (72.14±4.31 cps or 40.11±5.73 cps) (P<0.05), but higher than that of blastocysts frozned-thawed by controlled freezing method (34.41±3.32 cps and15.46±2.45 cps) (P<0.05).Experiment 4In this experiment two methods, HMC and microinjection, were imployed to produce equine-bovine iSCNT embryos, which equine fetal fibroblast cells was used as donor nuclei and bovine oocytes used as recipient cytoplasts. The level of CYBT gene encoded by the mitochondria in iSCNT embryos was detected by the quantitative RT-PCR. The results showed that: (1) the rates of fusion and cleavage of equine-bovine iSCNT embryos produced by HMC was significantly higher than that by microinjection. However there was no difference of blastocyst rates between these two methods. (2) the CYBT gene expression in early equine-bovine iSCNT embryos displayed heterogeneity. The equine CYBT gene expression had a gradually decreased trend from 1- cell stage to blastocyst period and the expression level at blstocyst stage had not been detected. The bovine CYBT copy number showed slow and gradual decrease trends from 1-cell to 8-cell stage, but the expression level sharply went up to the crest at 16-cell stage and increased to the 12-fold of that in 1-cell embryos, and then it decreased gradually at blastocyst stage.
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