| Intracytoplasmic sperm injection (ICSI) is a widespread used assisted reproductive technique, a kind of micro fertilization technology, and, often used in male infertility treatment as well as investigation of sperm-egg interaction. The aim of this study was to preliminarily investigate the pronucleus formation and the early embryonic development of heterologous zygotes generated by injecting of buffalo sperms into porcine oocytes, providing a reference data for future study in sperm-egg interaction of buffalo-pig sperm-egg interspecies.It was discussed in experiment 1 that the fertilization situation of buffalo sperm was injected into the porcine oocytes, and the results showed the pronucleus formation could be observed after the heterologous ICSI, although the pronucleus formation rate was significant lower than the homologous ICSI zygotes, i.e. pig sperms injected into the porcine oocytes (20.75% VS 38.60%, p<0.05). Cleavage rate of buffalo sperm injection group compared to that of sham injection and pig sperm injection group was not significant differences (78.20% VS 75.00% and 86.15%, p>0.05), but data of sham injection group was significant lower than that from pig sperm injected group (75.00% VS 86.15%, p>0.05). Presumptive blastocyst rate of sham injection compared to buffalo sperm injection and pig sperm injection group was not significant differences (28.79% VS 22.56% and 34.62%, p>0.05), however, buffalo sperm injection group was significantly lower than the pig sperm injection group (22.56% VS 34.62%, p<0.05). And blastula cell count of sham injection group, pig sperm and buffalo sperm group were not significant differences (53.80±10.60 VS 54.90±9.89 VS 54.75±10.98,p>0.05).In experiment 2, the effect of different cultivation system on buffalo-pig heterologous ICSI zygotes was investigated. The results showed that, the presumptive blastocyst rate of PZM-3 group was significantly higher than that of SOF full-course as well as cultured in PZM-3 for 2 d then changed to cultured in SOF (defined as P-SOF group) (18.63% VS 2.94% and 1.00%,28.30% VS 6.73% and 1.98%, respectively; p<0.05), regardless of the buffalo sperm or pig sperm injected.The objective of Experiment 3 was to investigate the effect of 5-Azacytidine (5-AzaC) treatment on the development of buffalo-pig heterologous ICSI eggs. The results showed that by treating buffalo sperm with 10 uM 5-AzaC for 12 h before injection, the pronucleus formation rate was not significant differences when injection eggs cultured in PZM-3 compared to culture in PZM-3 added with 10μM 5-AzaC (defined as 5A-PZM group) (15.69% VS 9.09%, p>0.05); Similarly, with untreated buffalo sperm injected into pig oocytes, the pronucleus formation rate of the PZM-3 group was not significant differences compared to the 5A-PZM group (11.11% VS 8.77%, p>0.05). As for the cleavage rate and blastocyst rate, when cultured in PZM-3, cleavage rate of treated buffalo sperm group significantly higher than that of untreated group (91.53% VS 82.44%, p<0.05), presumptive blastocyst rate was not significant differences (33.9%VS 31.30%,p>0.05). In addition, the blastula cells count, cultured in PZM-3 and 5A-PZM of group that buffalo sperm treated with 5-AzaC and untreated group was not significant difference (52.35± 10.40 VS 49.15 ± 11.96 and 51.70±9.75 VS 44.40±7.39, respectively; p>0.05).These above presented results suggested that:(1) Male pronucleus formation can be observed when buffalo sperm injected into the pig oocytes; (2) Buffalo - pig heterologous ICSI can provide theoretical reference for sperm quality evaluation; (3) Pig oocytes could be used in the research of heterologous fertilization with buffalo sperm. |
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