Proteomic Analysis Of The Differences Among Fresh And Post-thawed Boar Sperm, And Post-thawed Bull Sperm

There is a great difference between bull sperm and boar sperm in anti-freeze capability. The chemical composition and structure of the sperm have changed, the change of sperm proteins is considered to be one of the key factors that affect semen freezing efficiency. The main objective of the present studies was to analyze the differential proteomics among fresh boar sperm, post-thawed boar sperm and post-thawed bull sperm, and make a preliminary discussion on the dominant factor which causes the lower quality in the post-thawed boar sperm. It has all-important academic value and instructional significance on developing new frozen diluents and improving the quality of the cryopreserved sperm.In the studies, the protein which were from fresh boar sperm, cryopreserved boar and bull sperm were focused on7-cm immobilized pH gradient (linear pH:3-10) strip (IPGS), and then IPGS was electrophoresised on the sodium dodecyl sulfate polyacrylamide gel (SDS-PAG). After SDS-PAG was stained with coomassie blue R-250, differential proteins were detected by PDQuest8.0software and picked up with EXQuestTM Spot Cutter. Mass spectrometry of the protein was analyzed by matrix assisted laser desorption ionization/time of flight tandem mass spectrometry (MALDI-TOF-MS/MS) analyzer, and the protein were identified.The results show that boar sperm quality had a significant change during cryopreservation. The sperm motility, viability and membrane integrity of the post-thawed boar semen was significantly lower than the fresh boar semen (P<0.01). Moreover, the sperm quality of the post-thawed boar semen was significantly lower than post-thawed bull semen (P<0.01).In addition, the protein concentration of the boar sperm decreased significantly after freezing and post-thawing (P<0.01), which was lower than that of the post-thawed bull sperm. The molecular weight (MW) of proteins of the post-thawed bull sperm mainly distributed in the range of10-17kD,25-28kD and75-150kD, and proteins of fresh and post-thawed boar sperm distributed in range of10-17kD and25-75kD. The results suggested that the post-thawed bull sperm had more macromolecular proteins. The proteins content of post-thawed boar sperm were more than fresh boar sperm in range of10-17kD, meanwhile, which were significantly lower in range of25-75kD (P<0.05). The results suggested that Freezing and post-thawing might lead to the loss or degradation of the MW of25-75KD protein.The proteome of fresh boar sperm and post-thawed boar sperm had nine differential proteins, fresh boar sperm and post-thawed bull sperm had fifteen different proteins. The number of different proteins was four between post-thawed boar sperm and post-thawed bull sperm. A search according to the protein sequences in the national center for biotechnology information databases indicated that the differential proteins match to five corresponding proteins:mitochondrial F1-ATP synthase or mitochondrial a-ATP synthase, GRIN1(G protein-regulated inducer of neurite outgrowth1-like), ACRBP (acrosin-binding protein), ODF2(outer dense fiber of sperm tails2), and MDH (mitochondrial malate dehydrogenase). After cryopreservation of the boar sperm, mitochondrial F1-ATP synthase or mitochondrial a-ATP synthase, GRIN1and ACRBP had a significant reduction (P<0.05). Compared with the fresh boar sperm, post-thawed bull sperm had a higher quantity in ODF2and MDH.These studies suggested that there was a significant difference in certain protein from boar fresh and post-thawed, and bull spermatozoa, and sperm proteins might have degradation or leakage during the boar cryopreservation or post-thaw. Maybe, the differences can affect the effect of post-thawed boar sperm and be one main factor which causes the lower quality in the post-thawed boar sperm. The results provided an important evidence for further studies on cryodamage mechanism and proteomics of boar sperm. Furthermore, proteomic difference of the post-thawed sperm of boar and bull might reveal the reason that the quality of post-thawed boar sperm was lower than that of bull’s.