| There are many problems with buffalo's reproduction, such as delayed sexual maturation, unobvious estrus, long interim between estruses, low conception rate. The researches concerning embryo biotechnologies on buffalo are lagged behind that on cattle, and the reports about it are also relatively fewer. Although the success of program freezing of buffalo embryo had been reported and gave birth to children, there's no report about program freezing of buffalo oocytes. This study was to explore program freezing conditions for buffalo GV oocytes, aiming at improving protective effect of program freezing, to provide experimental materials for buffalo's IVM, IVF, nuclear transfer and other biotechnologies.Program freezing method was applied in this experiment, according to discussing crystal changes of several different freezing medium under different seeding temperatures, the freezing and protective effects of different cryopreservation medium on buffalo GV oocytes at different seeding temperatures were tested.1. During the experiments, the ovaries from cattle and buffalo were compared, the results indicated the number of oocytes from one cattle ovary is 13.4 on average, and that from one buffalo ovary was 4.85 on average. The latter was lower than the former, the diffrence was significant (p<0.01). The normal rate of oocyte configuration between cattle and buffalo was significantly different (p<0.01), respectively 87.9% and 66.3%.2. Seeding at -5.5℃,-6.5℃,-7℃ and using PBSm as control, the crystal status of PEGF, PEF, PGF at different time after seeding (2, 5, 8, 10, 15min) were observed.The results showed that PBSm completely rimmed in a short time after seeding (2min); when the seeding temperature came to -5.5℃, PEGF, PEF, PGF did not completely rime 10min after seeding, PGF did not completely rime 15min after seeding; When -6℃, PEGF, PEF, PGF did completely rime lOmin after seeding; When -6.5X:, PEGF, PEF, PGF completely rimed 8min after seeding; When -7℃, PEGF, PEF, PGF completely rimed 5min after seeding. And this demonstrated the crystal speed solely adding GL was lower than that solely adding EG, the Crystal effect of PEGF and PEF was better than that of GL and PBSm. At four seeding temperature, the crystal effects increased as seeding temperature decreased.3. Seeding at -6℃, -6.5℃, -7℃, the rate of oocytes with normal morpha after freezing and thaw-freezing was: PEGF (84%, 88.37%, 90.9%)>PEF (73.7%, 78%, 76.7%) > PGF (58%, 59%, 57.5%) > PBSm (50.7%, 47.2%, 47.4%). At the same seeding temperature, there was different between PEGF and PGF(p<0.05), between PEF and PBSm (p<0.05); and significance difference between PEGF and PBSm (p<0.01). When respectively seeding at -6℃, -6.5℃ and -7℃, thae effect of the same cryopreservation medium showed no difference.4. The survival rate of oocytes dyed by Trypan blue was: PEGF (76.9%, 87.5%, 83.3%) > PEF (64.0%, 70.8%, 66.7%) > PGF (47.4%, 55.0%, 55.6%) > PBSm (16.7%, 37.5%, 35.3%). The survival rate of oocytes was lower at -6℃ seeding than at -6.5℃ and -7℃ seeding. At these three seeding temperatures, the survival rate respectively between PEGF and PEF, PEF and PGF, PGF and PBSm was not different, but the survival rate respectively between PEGF and PGF, PEF and PBSm was significant different (p<0.01).5. Respectively seeding at -6℃,-6.5℃,-7℃, the maturation rate was : PEGF (18.9%, 20.5%, 20.0%) > PEF(9.7%, 18.2%, 15.6%) > PGF(6.9%, 7.7%, 8.3%) > PBSm(0, 0, 0). With regard to oocytes with normal morpha, after seeding at these three temperatures , PEGF and PEF, PEF and PGF were respectively not different ,but PEGF and PGF were different (p<0.05).after seeding at -6.5℃,two oocytes with PEGF as cryopreservation medium cleavaged ;one oocyte with PEF as cryopreservation medium cleavaged; one oocyte with PEGF as cryopreservation medium cleavaged after seeding at -7℃.In terms of these evidences ,the buffalo GV oocytes' freezing effect using PEGF as cryopreservation medium was good when seeding at -6.5℃ or -7℃.6. For... |
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