| Mammalian oocytes in follicule are arrested for a prolonged period at the diplotene stageof the first meiotic prophase. During follicullar growth, oocytes inatiate development. Whenreleased from follicle, fully gown oocytes can resume meiosis spontaneously and completenucleus and cytoplasmic maturation, which are essential for oocyte maturation. Oocytesachieve successful fertilization and embryonic development only when both of them arematured. Matured Oocytes arrested at metaphase II (MII) stage can resume meiosis untilstimulation occur. If fertilization or spontaneous activation is delayed, the matured oocyteswill undergo progressive aging. Aging decreases the potential of oocytes for fertilization andembryo development because of molecular changes occurring in the aged ooplasm.In present study, goat oocytes were studied through culture volume, maturation (nuclearand cytoplasm) and aging using various methods. The results are showed as follows:1. Under the culture proportion of one COCs/4μL medium, the maturation rate werestudied by different culture volume(50,100,250,500μL and1000μL). The results showedthat oocytes after in vitro culture, the maturation rate in100μL and250μL group were78.10%and74.62%respectively. There were no statistical difference between them butsignificantly higher than others (P <0.05). The results of parthenogenetic developmentshowed that the cleavage rate and blastocyst rate of100μL group were significantly higherthan others except250μL group2. The nuclear maturation was estimated for oocytes through chromosome stain. Ourresults showed that most of oocytes (80.87%) were arrested at GV stage before culture. Alongwith in vitro culture, oocytes resumed meiosis increasingly and complete nuclear maturation.At24h of culture, the proportion of oocytes at MⅡstage increased to91.50%(P <0.05).Subsequently, the proportion of oocytes at MⅡstage increased with no significant difference(P>0.05).3. The cytoplasmic maturation of goat oocytes was analysed by cortical granules (CGs)stain and ultrastructural study. The results of CGs stain showed that incomplete CGsmigration were found in all oocytes before culture. Along with the extended culture, the rateof oocytes with complete CGs migration increased significantly (66.39%,20h;85.05%,24h)(P <0.05). At28h, there was an increase for oocytes with complete CGs migration but nosignificant differences compared to24h. Ultrastructure of oocytes were studied bytransmission electron microscopy. The results showed that the morphological variation ofoocyte and the distribution and pattern of organelles in cytoplasm were consistent with cytoplasmic amturation.4. Oocytes aging were studied through FDA-PI double fluorescent dyes and cometassay. The results showed that the aged oocytes maitained at a low level by the perfectviability of membrane and little damaged DNA. Oocytes became aging gradually, along with theextended culture. Especially for30h, oocytes were seriously aged according to the decreased permeabilityof membrane and serious damaged DNA.5. Before24h culture, the cleavage rate and blastocyst rate of parthenogenetic oocytesincreased significantly with the extended culture, and reached to80.70%and29.15%(P <0.05). With the further culture of oocytes, the cleavage rate maitained at a higher level with nosignificant difference (P>0.05). The blastocyst rate decreased significantly with the extendedculture (P <0.05)except a slight increase at26h.6. In conclusion, under the culture density of one COCs/4μL medium, the culturevolume from100to250μL was better for oocytes maturation. Oocytes from antral follicles(2-6mm) could resume meiotic development during IVM. Most of oocyts could completenuclear and cytoplasmic maturation for24h of culture. Matured oocytes could become agedincreasingly with the extended culture, and the proportion of aged oocytes increasedsignificantly and the developmental ability decreased especially after culturing for24h later,... |
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