title: Sperm cryodamage occurs after rapid freezing phase: flow cytometry approach and antioxidant enzymes activity at different stages of cryopreservation.
abstract: BACKGROUND
  In order to improve the efficiency of bovine sperm cryopreservation process, it is important to understand how spermatozoa respond to differences in temperature as well as the ability to recover its own metabolism. The combination between flow cytometry approach and antioxidant enzymes activity allows a more sensible evaluation of sperm cell during cryopreservation. The aim of this study was to evaluate sperm attributes and antioxidant enzymes activity during different stages of cryopreservation process. Semen samples from Holstein bulls (n = 4) were separated in 3 treatments: fresh (37 °C); cooled (5 °C); and thawed. Evaluation occurred at 0 h and 2 h after incubation. Membrane integrity, mitochondrial membrane potential (MMP) and DNA damages were evaluated by flow cytometry; activities of antioxidant enzymes such as catalase, superoxide dismutase and gluthatione peroxidase were measured by spectrofotometry.
  
  
  RESULTS
  There was an increase in the percentage of sperm with DNA damage in the thawed group, compared to fresh and cooled, and for 2 hs of incubation when compared to 0 h. Considering MMP, there was an increase in the percentage of cells with medium potential in thawed group when compared to fresh and cooled groups. Opposingly, a decrease was observed in the thawed group considering high mitochondrial potential. Also in the thawed group, there was an increase on cells with damaged acrosome and membrane when compared to fresh and cooled groups. Significant correlations were found between antioxidant enzymes activity and membrane or mitochondrial parameters.
  
  
  CONCLUSIONS
  Based on our results, we conclude that cryopreservation affects cellular and DNA integrity and that the critical moment is when sperm cells are exposed to freezing temperature. Also, our study indicates that intracellular antioxidant machinery (SOD and GPX enzymes) is not enough to control cryodamage.
authors.affiliation: Laboratory of Spermatozoa Biology, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil.
authors.forename: L S
authors.initials: LS
authors.lastname: Castro
authors.affiliation: Laboratory of Spermatozoa Biology, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil.
authors.forename: T R S
authors.initials: TR
authors.lastname: Hamilton
authors.affiliation: Laboratory of Spermatozoa Biology, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil ; Laboratory of In Vitro Fertilization, Cloning and Animal Transgenesis, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil.
authors.forename: C M
authors.initials: CM
authors.lastname: Mendes
authors.affiliation: Laboratory of Andrology. Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil.
authors.forename: M
authors.initials: M
authors.lastname: Nichi
authors.affiliation: Laboratory of Andrology. Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil.
authors.forename: V H
authors.initials: VH
authors.lastname: Barnabe
authors.affiliation: Laboratory of Spermatozoa Biology, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil ; Laboratory of In Vitro Fertilization, Cloning and Animal Transgenesis, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil.
authors.forename: J A
authors.initials: JA
authors.lastname: Visintin
authors.affiliation: Laboratory of Spermatozoa Biology, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil.
authors.forename: M E O A
authors.initials: ME
authors.lastname: Assumpção
keywords: Bovine; DNA integrity; JC-1; Sperm viability