Construction Of Suppressive Subtractive Library Of Bovine Frozen And Fresh Sperm

The cryopreservation technique of mammalian spermatozoa plays an important role on animal husbandry industry.Through this technique,the utilization efficiency of excellent herd sire was improved,the quantity and feeding cost of herd were decreased greatly,and the genetic foundation was also propagated rapidly of the excellent herd sire by union breeding.And it also plays very important role on the preservation rare genetic resources.But now,the problems, such as low concepting rate,are also existed on spermatozoon cryopreservation.Spermatozoa are damged on the process of sperm freezing resuscitation,lead to sperm motility low.the effects of the diluent,frezzing rate and package type on the sperm acrosome and membrane structure were studied deeply by many scholars,But there is no satisfied study result.Uncover the cryodamage mechanism of spermatozoa limits the further improve of cryopreservation efficiency.As a genetic information transfer intermediate,transcripts play an important role on the process of spermatogenesis,sperm viability and sperm-oocyte binding.Through study on the transcriptomics of sperm systematically,the gene expression variance on the process sperm freezing,the evidence of sperm cryodamage,can be preliminary determined.But as terminal cell on the process of spermatogenesis,the morphology of mammalian spermatozoon was concentrated hardly,and there is only very thin layer membrane on external nucler,and with very lesscytoplasm and very little RNA.It is really a challenge to study the sperm transcripts.According to the structure and content characteristics,a bovine spermatozoa RNA extraction method,heat TRIzol method,was found and the bovine sperm RNA was extracted and detected by PCR analysis with an housekeeping gene GAPDH.The detected result indicated that the integrity tRNA was successfully extracted from bovine spermatozoa.And the quantity of the fresh and frozen sperm tRNA was amplified in the same scale with super smart technique, After amplification the double strands cDNA were digested with restriction enzyme RsaⅠ.The digested cDNA were divided into two parts,and the digested test cDNA were connected with different joint.The test cDNA with different joint were experienced continuous two times hybridization,then continuous twice PCR amplification for hybridization product were done, and the second PCR products was inserted into pGEMTeasy vector,then transformated into DH5αescherichia coli competence cell.After white/blue dot screening,1248 and 672 positive clones were obtained in positive and reverse library respectively.300 clones were picked randomly from positive library and negative library respectively.600 clones was sequenced and blast in the internet with the blast software against the data base.As a result,there is no homology gene in data for most of sequences.It may be bovine EST data are not completely. Tough study on new EST continuously,it will enrich database.It may discovery some of new cryodamge related transcripts.It provides test and theory foundation for sperm cryodamage mechanism.