Despite widespread application of intracytoplasmic sperm injection (ICSI) in human-assisted reproductive techniques (ART), the efficiency of this method is still far from satisfactory in livestock, particularly in the bovine species with its unique sperm condensation. On the basis of the natural chemical structure of chromatin in condensed sperm, we developed a novel combined heparin-reduced glutathione (GSH) sperm pretreatment that improves the efficiency of bovine ICSI via selection of the most appropriate sperm at the time of ICSI. Assessment of sperm DNA integrity revealed that this pretreatment can be considered as a safe and efficient approach for in vitro sperm decondensation when compared to conventional sperm pretreatments with dithiothreitol (DTT). Injection of completely decondensed bull sperm derived from this pretreatment significantly improved fertilization and blastocyst formation rates compared to untreated or intact sperm injection (34.8 ± 2.7 and 29.1 ± 1.5 vs. 12.0 ± 3.2 and 15.9 ± 1.2%, respectively; p<0.05). Real-time PCR analysis revealed that expression of pluripotent and anti-apoptosis markers in blastocysts derived by injection of completely decondensed sperm from heparin-GSH pretreatment were comparable to IVF when compared to the DTT pretreatment and control ICSI groups (p<0.05). The results of this study suggested that the degree of sperm decondensation derived from heparin-GSH pretreatment may affect ICSI efficiency in bovine.
In relation to the growing recent interest in the establishment of sperm-mediated gene transfer (SMGT) technology as a convenient and effective method for the simple production of transgenic animals, in this study the possibility of using SMGT to produce transgenic caprine embryos was investigated for the first time. Buck sperm were directly incubated with different concentrations (0-500 ng) of pcDNA/his/Lac-Z plasmid and used for IVF or ICSI. Sperm used for ICSI were categorized into motile or live-immotile group before being injected into oocytes. In a separate experiment, dead sperm prepared by repeated freezing/thawing were used for DNA-incubation before ICSI. Sham injection was carried out by intracytoplasmic injection of approximately the same volume of media containing different doses of DNA using an ICSI needle. Transgene expression and transmission were detected by X-Gal staining and PCR analysis of developed embryos, respectively. A reasonable blastocyst rate was observed in all the groups. Only embryos in the sham group were negative for transgene transmission. Transgene expression was completely dependent on the delivery technique and status of sperm, and was only observed in the live-immotile and dead ICSI groups. The results of this study showed that the technique (IVF vs. ICSI vs. sham injection), sperm status (motile vs. live-immotile vs. dead) and to some extent DNA concentration affect embryo development, transgene transmission and expression.
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