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Influence of seminal plasma during different stages of bovine sperm cryopreservation
Authors:Gabriela Bertaiolli Zoca  Eneiva Carla Carvalho Celeghini  Guilherme Pugliesi  Carla Patricia Teodoro de Carvalho  Mayra Elena Ortiz D'Avila Assumpção  Adriano Felipe Perez Siqueira  Leticia Zoccolaro Oliveira  Renata Lançoni  Rubens Paes de Arruda
Institution:1. Laboratory of Semen Biotechnology and Andrology – Center of Biotechnology in Animal Reproduction, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil;2. Laboratory of Teaching and Research in Pathology of Reproduction – Center of Biotechnology in Animal Reproduction, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil;3. Laboratory of Physiology and Molecular Endocrinology, Center of Biotechnology in Animal Reproduction – Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil;4. Laboratory of Sperm Biology, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil;5. Laboratory of Animal Reproduction, Department of Veterinary Clinics and Surgery, Veterinary School, University Federal of Minas Gerais, Belo Horizonte, Brazil
Abstract:This study aimed to evaluate the effect of seminal plasma on bovine sperm cryopreservation and to assess the integrity of plasma and acrosomal membranes, mitochondrial potential, remodelling of F-actin cytoskeleton and sperm chromatin fragmentation during the cooling, equilibrium and freezing/thawing stages. Six ejaculates collected from seven Nelore bulls (n = 42) were used in this study. Each ejaculate was divided into two aliquots (with seminal plasma = SP group; without seminal plasma = NSP group) and packed to a final concentration of 50 × 106 sperm per straw. Statistical analyses were performed using SAS software (version 9.3), and p ≤ .05 was considered significant. A time effect was observed for all sperm characteristics (p < .05), except for chromatin fragmentation (p > .05). The presence of seminal plasma better preserved the acrosomal integrity (SP = 75.2% and NSP = 71.7%; p < .05) and also provided lower F-actin remodelling during cryopreservation process (SP = 29.9% and NSP = 32.4%; p < .05). Regarding to the cryopreservation stages, it was observed that cooling step induced higher remodelling of F-actin than the equilibrium and freezing/thawing stages (56.3%, 32.2% and 23.9%, respectively; p < .05). The equilibrium step had minor influence on overall sperm characteristics while the freezing/thawing stage was responsible for the highest percentage of damage in plasma membrane (?65.2%), acrosomal membrane (?34.0%) and mitochondrial potential (?48.1%). On the other hand, none of the cryopreservation stages affected chromatin integrity. It was concluded that the presence of seminal plasma provides increased acrosomal integrity and reduced remodelling of F-actin cytoskeleton. Higher F-actin remodelling is observed after the cooling step while the freezing/thawing step is most damaging to sperm membranes and mitochondrial potential during bovine sperm cryopreservation.
Keywords:bull  chromatin  cryopreservation stages  F-actin cytoskeleton  seminal plasma
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