Effects Of Nutrition Factors On Development In Vitro Of Cow Oocytes And Cloning Efficiency

Cow cloning by somatic cell nuclear transfer (SCNT) has the potential to improve the productivity of enormously valuable livestock. It also has the potential for producing transgenic animals that synthesize large quantities of human proteins for use in medicine. However, low efficiency and high abortion rate and early developmental abnormity of offspring were bottlenecks of cloning application. The success of SCNT techniques depends mainly on two factors including oocyte maturation and its activation. Incomplete oocyte maturation, especially cytoplasmic maturation, and incomplete oocyte activation may result in imprecise reprogramming. Therefore, improvement in media for oocyte maturation and optimization of activation protocol become two pivotal processes for considerable improvement in cloning efficiency.Therefore, a serum-free chemically defined maturation media and activation protocols of cow oocytes were optimized in the present study. The SCNT were performed to confirm the feasibility of optimized maturation media and activation protocol taking cow ear fibroblast, oviduct epithelial cells and cumulus cells as nuclear donors. Effects of GTPs on preimplantation development of IVF and NT embryos and its mechanism were also investigated.1. Culture of donor cells for cow cloningThe fibroblasts were separated from cow ear tissue and were cultured in DMEM/F12 media with serum. Oviduct epithelial cells were separated by mechanical method and cultured in the same media as fibroblast. Cumulus cells were separated from cumulus-oocyte complexes (COCs) by trypsin digestion and cultured in the optimized serum-free McCoy's5a media. Morphological characteristics, growth curves and karyotypes were analyzed after passage and purification of three types of cells. The results showed that the morphological characteristics, growth curves and karyotypes of three types of cells were normal and accorded with growth rules of common cells. Three types of cells could be used as donor in the cloning technique.2. Optimization of serum-free media for cow oocytes maturationA chemical defined basic medium have been constituted by adding PVP-40 instead of serum. The different concentrations of glucose (0 mM,1.5mM,5.6 mM and 20 mM), IGF-I (0 ng/mL,50 ng/mL,100 ng/mL and 150 ng/mL) and GTPs (0μM,10μM,15μM and 20μM) added into the basic medium, respectively. The oocytes were cultured in these medium with glucose, IGF-I and GTPs, respectively. The results showed that the concentration of glucose, IGF-I and GTPs with significantly higher cleavage and blastocyst rates were 5.6 mM,100 ng/mL and 15μM, respectively. Then three substances with the above concentration were added together into basic medium and constituted an optimized medium (Optimized SOFaa), which was serum-free and chemical defined. Oocytes were cultured in control TCM-199+FBS media and optimized media, respectively. The results showed that optimized media cans instead TCM-199+FBS media as cow oocytes maturation. The rate of development to blastocyst of oocytes maturation with optimized media was significant higher than those maturation with control TCM-199+FBS media (39.2% vs.30.4%, P＜0.05). This improvement in development capacity of preimplantation embryos contributes to the increase of intracytoplasmic glutathione concentration (14.3 pmol/oocyte vs.8.9 pmol/oocyte, P＜0.05). The present study also approved that oocytes collected by puncture method from 3-6mm follicles in autumn had higher the number of oocyte per ovary, rate of maturation, cleavage and development to blastocyst.3. Optimization of activation methods of cow oocytesThe activation methods of cow oocytes were optimized through comparing the efficiency of different treatments on the activation and subsequent development of oocytes. Cumulus-free oocytes which matured with optimized SOFaa were activated according to the experiment designs. For activation treatment alone, the matured oocytes were treated with single activation agents including ionomycin (5μM for 5 min), ethanol (7% for 7 min), calcium ionophore A23187 (5μM for 5 min), or strontium (10 mM for 5 h). The pronuclear formation (39.4%,41.2% vs.21.3%,25.2%) and cleavage rate (30.3%,28.1% vs.19.7%,23.7%) were higher significantly in ionomycin and ethanol treatment alone compared to other treatments (P< 0.05). For the combined activation treatment, the same concentrations of ionomycin and ethanol as above were used in combination with 6-dimethylaminopurine (6-DMAP,2.0 mM for 3 h) or cycloheximide (CHX)+cytochalasin B (CB, 10μg/mL for 3 h). The pronuclear formation (51.0%,53.4%vs.39.6%,35.3%), cleavage rate (41.3%,41.6%vs.28.1%, 26.1%) and blastocyst rate (18.2%,19.3%vs.10.1%,11.1%) were higher significantly (P< 0.05) in ionomycin+6-DMAP treatment and ethanol+CHX+CB treatment compared to other treatments. The parthenogenetic blastocysts produced by activation with ionomycin+6-DAMP and ethanol+CHX+CB and in vitro fertilized blastocysts (control group) were examined for apoptosis and ploidy. The ethanol+CHX+CB treatment shown lower significantly blastocyst apoptosis index (7.0%vs.9.1%, P< 0.05) and percentage of haploid (7.0%vs.9.1%, P< 0.05) compared to ionomycin+ 6-DAMP treatment. These results suggested that ethanol+CHX+CB treatment was more favorable method for parthenogenesis of bovine oocytes.4. Test of optimized maturation medium and activation method of cow oocytes in cloningTo examine the feasibility of the optimized SOFaa media and activation method in cow cloning, oocytes were matured with the optimized SOFaa media and control TCM-199+FBS media, respectively and then SCNT were performed taking cumulus cells as donors. The results showed that the optimized media may instead TCM-199+FBS media for maturation of recipient oocytes. The rates of NT embryos development to blastocysts were significant higher in oocytes maturation with optimized media than that maturation with control TCM-199+FBS media (21.2%vs. 14.3%, P< 0.05). The optimized activation protocol was also practicable in cow cloning. To test the effects of types of donors on preimplantation development of NT embryos, SCNT were performed taking ear fibroblasts, oviduct epithelial cells and cumulus cells as donors, respectively. The rates of NT embryos development to blastocysts were significant higher when cumulus cells as donors than when fibroblasts and oviduct epithelial cells as donors (21.2% vs.15.9%,13.7%, P<0.05). The cumulus cells were more favorable type for cow cloning. To examine the effects of cell cycle of donors on efficiency of cloning, the SCNT were done by using GO-stage and non-GO stage cumulus cells as donors respectively. The result indicated that when used GO-stage cells as donors, blastocyst rate was raised significantly (21.2% vs.12.2%, P<0.05).5. Effects of GTPs on development of IVF and NT embryos and approach its mechanismOocytes and embryos are inevitably more exposed to oxygen in vitro, which results in increased production of ROS than in vivo because of the necessary manipulations with transient exposure to atmospheric oxygen and visible light during embryos production by IVF and NT. In bovine, the detrimental effects of ROS to embryo development have been confirmed. Therefore, the addition of antioxidants to IVC media may be pre-requisite for improvement of development competence of embryos by in vitro production. The effects of GTPs on preimplantation development of IVF and NT embryos were investigated. The matured oocytes were fertilized and embryos were cultured in a defined conditioned media with or without GTPs supplementation (0,10,15,20,25μM). The results demonstrated supplementation with GTPs on different concentrations had no improvements in rate of cleavage and blastocyst formation (P＜0.05). Supplementation of the culture media of IVF embryos with 15μM GTPs improved rate of blastocyst formation (33.6% vs.24.8%, P< 0.05), while higher GTPs concentrations (25μM) significantly reduced embryos development (P<0.05). Effects of GTPs on NT embryos were the same as that on IVF embryos. This improvement in development capacity of preimplantation IVF and NT embryos contributes to the increase of intracytoplasmic glutathione concentration.