Study On The Technology Of Intracytoplasmic Sperm Injection In Porcine

Intracytoplasmic sperm injection(ICSI) is a new and important technique of in vitro fertiliztion, and it has succeed in many species, including porcine. With the development of transgenic animal and embryo engineering, many research focused on the ICSI of porcine. However, there are many factors limited the low efficiencies of ICSI in procine.The persent study investigated the effects of different methods on the embryonic development after ICSI through micromanipulation with Piezo, such as adding different components, different sperm pretreatment and different activation. In addition, we compared the embryonic development of the embryos derived from ISCI with frozen sperm and IVF. In Exp. 1: Sperm rendered inmotile by three-time freezing and thawing in liquid nitrogen, pre-incubated with 0.1% Triton X-100 and 50 k HZ ultrasonic concussion for 10 s in room temperature were designed as experiment group. In Exp. 2: After three-time freezing and thawing in liquid nitrogen, ICSI was performed in 100 ul TL-HEPES supplemented with 7.5ug/ml cytochalasin B or 5% PBS-PVA. No supplements in 100 ul TL-HEPES was designed as control group. In Exp. 3: After three-time freezing and thawing in liquid nitrogen, 2 DC pulses of 1.2kv/cm for 30 us, 6-DMAP for 6 h and Calcium ionophore A23187 for 5min. No activation was designed as control group. In Exp. 4: Base on the above results, we choosed the most efficiency method. After three-time freezing and thawing in liquid nitrogen, ICSI was performed in the TL-HEPES supplemented with CB and PVA following electric-activation. We compared the pronuclear formation rates and embryonic development between ICSI and IVF embryo. The main results were as following:In Exp. 1: The cleavage and blastocyst rates were significantly increased in the three pretreatment grounps(82.57%, 87.60%, 65.69% vs 57.59%; 33.33%, 33.67%, 35.48% vs 20.07%; P < 0.05). In Exp. 2: The micromanipulation survival rate of CB or PVA group were significantly higher than control(86.55% vs 78.45%; 85.96% vs 80.17%; P < 0.05). However, there were no significantly difference in the cleavage and blastocyst rates(87.38% vs 93.41%; 84.96% vs 89.25%; 30.39% vs 23.58%;32.76% vs 25.47%; P > 0.05). In Exp. 3: For the three activation methods, the electric-activation could significantly increased blastocyst rates compared to control group(24.37% vs 8.66%; P <0.05). In Exp. 4: The pronuclear formation, cleavage and blastocyst rates of ICSI groups were significantly higher than IVF group(42.59% vs 23.33%, 80.65% vs 69.83%;27.55% vs 17.17%; P < 0.05).These results showed that different sperm pretreatment could promote the cleavage and blastocyst rates, and the suppliment with CB or PVA have no significantly effect; The electric-activation could promote cleavage and blastocyst rates. Based these results, we compared the pronuclear formation rates and embryonic development between ICSI and IVF embryo using our optimal ICSI protocol, the results indicated that the cleavage rates and the cell number of blastocyst were same between ICSI and IVF groups; however, the blastocyst rate of ICSI was significantly higher than IVF. In addition, the karyotype analysis showed that our ICSI protocol could significantly promote the rates of normal karyotype. Our resuls together showed that our ICSI protocol can promote the efficiency of in vitro porcine embryo production.