title: A 31-kDa seminal plasma heparin-binding protein reduces cold shock stress during cryopreservation of cross-bred cattle bull semen.
abstract: In the present study, a 31-kDa protein, purified from cattle bull seminal plasma heparin-binding proteins (SP-HBP), was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. Raw semen of six cross-bred bulls was treated with 31-kDa HBP before cryopreservation to observe its effect on motility, viability, hypo-osmotic swelling test, acrosome integrity, in vitro capacitation/acrosome reaction, and oxidative stress at pre-freeze and frozen-thawed phases of cryopreservation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 31-kDa protein eluted and purified from SP-HBP (separated on acrylamide gels) resulted in a single band of 40 kDa. In matrix-assisted laser desorption/ionization-time of flight analysis, 12 peptides were identified with matching significantly (P < 0.05) to interlukin-6 of bovine with a top score of 55. Addition of 25 μg/mL of fluorescein isothiocyanate-conjugated 31-kDa protein to raw semen and incubation at 37 °C for 20 minutes before cryopreservation resulted in its binding mainly to head region. Treatment of semen with 31-kDa HBP resulted in a significant (P < 0.05) average increase of 9.2%, 6.8%, and 11.7% and 5.5%, 6.5%, and 11.0% in motile, viable, hypo-osmotic swelling-responsive spermatozoa in six bulls at pre-freeze and frozen-thawed phases of cryopreservation, respectively. Percentage of spermatozoa with intact acrosomes nonsignificantly enhanced in the semen treated with 31-kDa HBP at both phases of cryopreservation. An average nonsignificant increase of 3.1% in in vitro capacitated and acrosome-reacted spermatozoa was obtained in semen supplemented with 31-kDa HBP. Addition of 31-kDa HBP also nonsignificantly reduced Malonadialdehyde (MDA) level by 10.7 and 19.3 μM/10(9) spermatozoa in prefrozen and frozen-thawed semen, respectively. The results obtained here indicate to conclude that treatment of cross-bred cattle bull semen with 31-kDa HBP protects the spermatozoa from cold shock effect by coating the sperm surface.
authors.affiliation: Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India.
authors.forename: M K
authors.initials: MK
authors.lastname: Patel
authors.affiliation: Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India. Electronic address: ranjna.cheema@gmail.com.
authors.forename: R S
authors.initials: RS
authors.lastname: Cheema
authors.affiliation: Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India.
authors.forename: A K
authors.initials: AK
authors.lastname: Bansal
authors.affiliation: Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India.
authors.forename: V K
authors.initials: VK
authors.lastname: Gandotra
mesh_terms: D020101:Acrosome Reaction / Q000187:drug effects; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D002417:Cattle; D015925:Cryopreservation / Q000662:veterinary; D003451:Cryoprotective Agents; D006493:Heparin / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D018384:Oxidative Stress / Q000187:drug effects; D012661:Semen / Q000737:chemistry / Q000502:physiology; D012662:Semen Preservation / Q000662:veterinary*; D029589:Seminal Plasma Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D013075:Sperm Capacitation / Q000187:drug effects; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects / Q000502:physiology
chemical_list: D002352:Carrier Proteins; D003451:Cryoprotective Agents; D029589:Seminal Plasma Proteins; D006493:Heparin
keywords: 31 kDa; SP-HBP; cattle bull; cold shock stress; semen