|คำค้น||:||กระบือปลัก , การผลิตตัวอ่อนในหลอดแก้ว , การย้ายฝากนิวเคลียสด้วยเซลล์ร่ายกาย|
|อ้างอิง||:||http://elibrary.trf.or.th/project_content.asp?PJID=BRG5080026 , http://research.trf.or.th/node/6077|
This project was divided into 6 experiments. In Experiment 1, the objective of this study was to optimize the activation protocol for buffalo oocytes after intracytoplasmic sperm injection (ICSI). The release of the second polar body (2nd PB) at 3, 6 and 9 h after ICSI of in vitro matured oocytes activated either with 5μM ionomycin (Io) or 7% ethanol (EtOH) was preliminary examined. The highest rate of 2nd PB extrusion occurred at 3 h of activation and the 2nd PB extrusion in EtOH group (68%) was significantly higher than in Iogroup (46%). Oocytes that extruded the 2nd PB were selected and cultured either with 1.9 mM 6- dimethylaminopurine (6-DMAP) for 3 h or with 10 µg/mL cychloheximide (CHX) for 5 h. Significantly higher rate of oocytes formed male and female pronuclei in EtOH combined with CHX (EtOH+CHX) (62%) group compared to those of Io+CHX (42%) and EtOH+6-DMAP (48%) groups whereas Io+6-DMAP group showed intermediate value (58%). Significantly higher blastocyst formation rates were obtained in Io+6-DMAP (29%) and EtOH+CHX (24%) groups than in Io+CHX (6%) and EtOH+6-DMAP (17%) groups. Our results indicate that buffalo ICSI oocytes are effectively activated by combination treatment of Io with 6-DMAP and EtOH with CHX resulting in the highest cleavage and blastocyst formation rates. Experiment 2, the objective of this experiment was to investigate the effect of donor cell types on the developmental potential and quality of cloned swamp buffalo embryos in comparison with cloned cattle embryos. Fetal fibroblasts (FFs), ear fibroblasts (EFs), granulosa cells (GCs) and cumulus cells (CCs) were used as the donor cells in both buffalo and cattle. The cloned cattle or buffalo embryos were produced by fusion of the individual donor cells with enucleated cattle or buffalo oocytes, respectively. The reconstructed (cloned) embryos and in vitro matured oocytes without enucleation were parthenogenetically activated (PA) and cultured for 7 days. Their developmental ability to the blastocyst stage was evaluated. The total number of trophectoderm (TE) and inner cell mass (ICM) cells and the ICM ratio in each blastocyst was determined by differential staining as an indicator of embryo quality. The fusion rate of CCs with enucleated oocytes was significantly lower than for those of other donor cell types both in cattle and buffalo. The rates of cleavage and development to the 8 cell, morula and blastocyst stages of cloned embryos derived from all donor cell types did not significantly differ within the same species. However, the cleavage rate of cloned cattle embryos derived from FFs was significantly higher than those of cattle PA and cloned buffalo embryos. The blastocyst rates of cloned cattle embryos, except for the ones derived from CCs, were significantly higher than those of cloned buffalo embryos. In buffalo, only cloned embryos derived from CCs showed a significantly higher blastocyst rate than that of PA embryos. In contrast, all the cloned cattle embryos showed significantly higher blastocyst rates than that of PA embryos. There was no difference in ICM ratio among any of the blastocysts derived from any of the donor cell types and PA embryos in both species. FFs, EFs, GCs and CCs had similar potentials to support development of cloned cattle and buffalo embryos to the blastocyst stage with the same quality. Experiment 3, the objective of this experiment was to investigate the potential of swamp buffalo oocytes vitrified-warmed at the metaphase of the second meiotic cell division (M-II) stage to develop to the blastocyst stage after parthenogenetic activation (PA) or intracytoplasmic sperm injection (ICSI). In Experiment 1, we examined the effects of exposure time of oocytes to cryoprotectants (CPA) on their in vitro development after PA. In vitro matured(IVM) oocytes were placed in10% dimethylsulfoxide (DMSO)+ 10% ethylene glycol (EG) for 1 min and then exposed to 20% DMSO + 20% EG + 0.5 M sucrose for 30 s, 45 s or 60 s (1min+30s, 1min+45s and 1min+60s groups, respectively). The oocytes were then exposed to warming solution (TCM199 HEPES + 20% FBS and 0.5M sucrose) for 5 min and then washed in TCM199 HEPES + 20% FBS for 5 min. IVM oocytes without CPA treatments served as a control group. The viability assessed by fluorescein diacetate (FDA) staining was 100% in all groups. The developmental rates after PA to the blastocyst stage between 1min+30s (16%) and control (26%) groups did not differ significantly, but they were significantly higher than those in 1min+45s (10%) and 1min+60s (2%) groups. In Experiment 2, we examined the effect of two CPA exposure times, 1min+30s and 1min+45s on the in vitro development after PA of oocytes vitrified by the Microdrop method. The viabilities in vitrified 1min+30s, 1min+45s and the control (without CPA treatments) groups were not different (97%, 95% and 100%, respectively). The development of surviving oocytes to the blastocyst stage in the vitrified 1min+30s group (8%) was significantly higher than that in the vitrified 1min+45s group (4%) and significantly lower than those in control group (26%). In Experiment 3, we examined the effect of two CPA exposure times, 1min+30s and 1min+45s on in vitro development after ICSI of vitrified oocytes. Viabilities in vitrified oocytes among 1min+30s, 1min+45s and control groups were not different (96%, 91% and 100%, respectively). After ICSI, vitrified-warmed oocytes were activated and oocytes with the second polar body were cultured for 7 days. The development of ICSI oocytes to the blastocyst stage in the vitrified 1min+30s group (11%) was significantly higher than that in the vitrified 1min+45s (7%) group and significantly lower than those in control group (23%). In conclusion, our study demonstrated that the 1min+30s CPA treatment regimen could yield the highest blastocyst formation rates after PA and ICSI for oocytes vitrified by the Microdrop method. Experiment 4, the objective of this experiment was to examine the effects of two types of vitrification solution, and of two types of vitrification technique on the survival rate of vitrified-warmed buffalo MII oocytes were examined. Furthermore, the developmental capacity of vitrified-warmed buffalo MII oocytes following intracytoplasmic sperm injection (ICSI) was investigated. In vitro matured (IVM) oocytes were randomly separated into 6 groups and cryopreserved by using 1) Cryotop method combined with VA solution (10% DMSO+10% EG for 1 min and then 20% DMSO+20%EG+0.5M sucrose for 30 sec; Cryotop+VA), 2) Cryotop method combined with VB solution (4% EG for 12-15 min and then 35% EG+5% PVP and 0.4 M trehalose for 30 sec; Cryotop+VB), 3) Microdrop method combined with VA solution (Microdrop+VA), 4) Microdrop method combined with VB solution (Microdrop+VB), 5) Control oocytes stained with fluorescein diacetate (FDA) (control), 6) Fresh control oocytes without staining by FDA (fresh control). The survival rate of oocytes was examined by FDA staining and surviving oocytes were subjected to ICSI. The oocytes viability of VA solution (Microdrop+VA: 93% and Cryotop+VA: 97%) were significantly higher than that in VB solution (Microdrop+VB: 79% and Cryotop+VB: 81%), but significantly lower than that in control groups (100%). The 2nd PB extrusion rate and the capacity of embryo development to the blastocyst stage in control and fresh control groups were significantly higher than that in vitrified groups, but no difference among vitrified groups. There was no difference between control and fresh control groups in 2nd PB extrusion and embryo development. In conclusion, VA solution could yield higher survival rate of vitrified oocytes than VB solution, Cryotop and Microdrop are equally suitable techniques for buffalo oocytes vitrification. Experiment 5, the objective of this experiment was to investigate the effects of two types of vitrification solution and two types of vitrification method on the survival and developmental rates of matured swamp buffalo oocytes after in vitro fertilization (IVF). In vitro-matured oocytes were randomly divided into 5 groups and cryopreserved by using 1) Cryotop method combined with VA solution (10% DMSO+10% EG for 1 min and then 20% DMSO+20%EG+0.5M sucrose for 30 sec; Cryotop+VA), 2) Cryotop method combined with VB solution (4% EG for 12-15 min and then 35% EG+5% PVP and 0.4 M trehalose for 30 sec; Cryotop+VB), 3) SSV method combined with VA solution (SSV+VA), 4) SSV method combined with VB solution (SSV+VB), 5) Fresh oocytes without staining by FDA (fresh control). The survival rate of oocytes was examined by FDA staining and surviving oocytes were subjected to IVF. The survival rate of vitrified-warmed oocytes in Cryotop+VA (92%) group was significantly higher than that of SSV+VA (86%), Cryotop+VB (76%) and SSV+VB (71%) groups. However, they were still significantly lower than that of fresh control group (100%). The blastocyst rate of vitrified oocytes in Cryotop+VA group (9%) was significantly higher than that of Cryotop+VB (3%), SSV+VA (5%) and SSV+VB (1%) groups, but still significantly lower than that of fresh control group (19%). From these results can be concluded that Cryotop vitrification method combined with VA solution yielded the highest survival and blastocyst rates. Experiment 6, the objective of this experiment was to examine the effects of growth factors (IGF-I, EGF and bFGF) on in vitro growth of swamp buffalo early antral follicles. Buffalo early antral follicles were isolated from ovaries by enzymatic and mechanical approaches. The early antral follicles were divided into 3 groups, depended on their diameters, group I: 200-399 μm, group II: 400-599 μm and group III: 600-799 μm. The collagen-embedded follicles was cultured in in vitro growth (IVG) medium supplemented with growth factors by 8 treatments: Treatment 1: no supplementation of growth factors as a control, Treatment 2: 50 ng/mL basic fibroblast growth factor (bFGF), Treatment 3: 100 ng/mL insulin-like growth factor-I (IGF-I), Treatment 4: 50 ng/mL epidermal growth factor (EGF), Treatment 5: 50 ng/mL bFGF+100 ng/mL IGF-I, Treatment 6: 50 ng/mL bFGF+50 ng/mL EGF, Treatment 7: 100 ng/mL IGF-I +50 ng/mL EGF, Treatment 8: 8 50 ng/mL bFGF+100 ng/mL IGF-I+50 ng/mL EGF. The follicles were cultured in 4-well dishes for 14 days. The diameter of follicle was measured at day 7 and 14 of cultured. The results showed that the diameter of follicle at day 7 in group I (200-399 μm) that had been cultured in medium supplemented with bFGF, IGF-I, bFGF+IGF-I, bFGF+EGF, IGF-I+EGF and bFGF+IGFI+ EGF were able to increased follicle diameters but no significantly difference (P>0.05), whereas, at day 14, follicles in group I which cultured in medium supplemented with bFGF and bFGF+IGF-I were significantly increased their diameters (P<0.05). The results of follicle cultured in group II (400-599 μm) at day 7 showed that follicle which had been cultured in medium supplemented with bFGF+IGF-I had significantly higher increased follicle diameters than other treatments (P<0.05). However, at day 14, follicles that had been cultured in medium supplemented with bFGF were able to increased follicle diameters more than other treatments (P>0.05). The results of buffalo early antral follicles cultured in group II indicated that follicle which culture in medium supplemented with bFGF and bFGF+IGF-I were able to increase follicle diameters at day 7 and day 14 and this was higher enlargement than those in other treatments. The follicles in groups III (600-799 μm) that had been cultured in medium supplemented with bFGF, IGF-I and bFGF+IGF-I were able to increased follicle diameters at day 7 (P<0.05). On the other hand, at day 14, only follicles that had been cultured in medium supplemented with bFGF were able to increase follicle diameters (P<0.05). The result of FDA staining to examine oocytes viability showed that bFGF had higher percentage of viable oocytes than other treatments. Moreover, the culture time had an effect on follicle culture. The follicles in diameter of 200-399 μm required a longer culture time more than 14 day as showed in the result of meiotic stage which arrested at GV stage. Follicles in diameter of 400-599 μm were suitable for culture at 14 days. The result showed that meiotic stage was able to reach MI, whereas culturing follicles in diameter of 600-799 μm required less than 14 days to support the oocytes viability which were found large number of oocytes already lysis because of too long culture period. These results indicated that bFGF and IGF-I were able to improve the growth of early antral buffalo follicles whereas EGF is unable to do that. This work will be essential for in vitro cultivation of immature oocytes which can be used for other applications
รังสรรค์ พาลพ่าย . (2554). การอนุรักษ์และเพิ่มจำนวนกระบือปลักพันธุ์ดีด้วยการผลิตตัวอ่อนในหลอดแก้วและการย้ายฝากนิวเคลียสด้วยเซลล์ร่ายกาย.
กรุงเทพมหานคร : สำนักงานกองทุนสนับสนุนการวิจัย.
รังสรรค์ พาลพ่าย . 2554. "การอนุรักษ์และเพิ่มจำนวนกระบือปลักพันธุ์ดีด้วยการผลิตตัวอ่อนในหลอดแก้วและการย้ายฝากนิวเคลียสด้วยเซลล์ร่ายกาย".
กรุงเทพมหานคร : สำนักงานกองทุนสนับสนุนการวิจัย.
รังสรรค์ พาลพ่าย . "การอนุรักษ์และเพิ่มจำนวนกระบือปลักพันธุ์ดีด้วยการผลิตตัวอ่อนในหลอดแก้วและการย้ายฝากนิวเคลียสด้วยเซลล์ร่ายกาย."
กรุงเทพมหานคร : สำนักงานกองทุนสนับสนุนการวิจัย, 2554. Print.
รังสรรค์ พาลพ่าย . การอนุรักษ์และเพิ่มจำนวนกระบือปลักพันธุ์ดีด้วยการผลิตตัวอ่อนในหลอดแก้วและการย้ายฝากนิวเคลียสด้วยเซลล์ร่ายกาย. กรุงเทพมหานคร : สำนักงานกองทุนสนับสนุนการวิจัย; 2554.