title: Novel Flow Cytometry Analyses of Boar Sperm Viability: Can the Addition of Whole Sperm-Rich Fraction Seminal Plasma to Frozen-Thawed Boar Sperm Affect It?
abstract: Boar semen cryopreservation remains a challenge due to the extension of cold shock damage. Thus, many alternatives have emerged to improve the quality of frozen-thawed boar sperm. Although the use of seminal plasma arising from boar sperm-rich fraction (SP-SRF) has shown good efficacy; however, the majority of actual sperm evaluation techniques include a single or dual sperm parameter analysis, which overrates the real sperm viability. Within this context, this work was performed to introduce a sperm flow cytometry fourfold stain technique for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential. We then used the sperm flow cytometry fourfold stain technique to study the effect of SP-SRF on frozen-thawed boar sperm and further evaluated the effect of this treatment on sperm movement, tyrosine phosphorylation and fertility rate (FR). The sperm fourfold stain technique is accurate (R2 = 0.9356, p > 0.01) for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential (IPIAH cells). Centrifugation pre-cryopreservation was not deleterious (p > 0.05) for any analyzed variables. Addition of SP-SRF after cryopreservation was able to improve total and progressive motility (p < 0.05) when boar semen was cryopreserved without SP-SRF; however, it was not able to decrease tyrosine phosphorylation (p > 0.05) or improve IPIAH cells (p > 0.05). FR was not (p > 0.05) statistically increased by the addition of seminal plasma, though females inseminated with frozen-thawed boar semen plus SP-SRF did perform better than those inseminated with sperm lacking seminal plasma. Thus, we conclude that sperm fourfold stain can be used to simultaneously evaluate plasma and acrosomal membrane integrity and mitochondrial membrane potential, and the addition of SP-SRF at thawed boar semen cryopreserved in absence of SP-SRF improve its total and progressive motility.
authors.affiliation: Laboratory of Andrology and Technology of Swine Embryos, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga, São Paulo, Brazil.
authors.forename: Mariana Andrade
authors.initials: MA
authors.lastname: Torres
authors.affiliation: Center of Excellence in Biotechnology of Reproduction, University of La Frontera, Temuco, Araucania, Chile.
authors.forename: Rommy
authors.initials: R
authors.lastname: Díaz
authors.affiliation: Center of Excellence in Biotechnology of Reproduction, University of La Frontera, Temuco, Araucania, Chile.
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Boguen
authors.affiliation: Laboratory of Andrology and Technology of Swine Embryos, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga, São Paulo, Brazil.|Laboratory of Swine Research, Department of Animal Nutrition and Production, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga, São Paulo, Brazil.
authors.forename: Simone Maria Massami Kitamura
authors.initials: SM
authors.lastname: Martins
authors.affiliation: Laboratory of Andrology and Technology of Swine Embryos, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga, São Paulo, Brazil.
authors.forename: Gisele Mouro
authors.initials: GM
authors.lastname: Ravagnani
authors.affiliation: Laboratory of Andrology and Technology of Swine Embryos, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga, São Paulo, Brazil.
authors.forename: Diego Feitosa
authors.initials: DF
authors.lastname: Leal
authors.affiliation: Laboratory of Andrology and Technology of Swine Embryos, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga, São Paulo, Brazil.
authors.forename: Melissa de Lima
authors.initials: Mde L
authors.lastname: Oliveira
authors.affiliation: Laboratory of Andrology and Technology of Swine Embryos, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga, São Paulo, Brazil.
authors.forename: Bruno Bracco Donatelli
authors.initials: BB
authors.lastname: Muro
authors.affiliation: Laboratory of Andrology and Technology of Swine Embryos, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga, São Paulo, Brazil.
authors.forename: Beatriz Martins
authors.initials: BM
authors.lastname: Parra
authors.affiliation: Department of Veterinary Medicine, School of Animal Sciences and Food Engineerig, University of São Paulo, Pirassununga, São Paulo, Brazil.
authors.forename: Flávio Vieira
authors.initials: FV
authors.lastname: Meirelles
authors.affiliation: Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.
authors.forename: Frederico Ozanan
authors.initials: FO
authors.lastname: Papa
authors.affiliation: Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.
authors.forename: José Antônio
authors.initials: JA
authors.lastname: Dell'Aqua
authors.affiliation: Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.
authors.forename: Marco Antônio
authors.initials: MA
authors.lastname: Alvarenga
authors.affiliation: Laboratory of Swine Research, Department of Animal Nutrition and Production, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga, São Paulo, Brazil.
authors.forename: Aníbal de Sant'Anna
authors.initials: Ade S
authors.lastname: Moretti
authors.affiliation: Center of Excellence in Biotechnology of Reproduction, University of La Frontera, Temuco, Araucania, Chile.
authors.forename: Néstor
authors.initials: N
authors.lastname: Sepúlveda
authors.affiliation: Laboratory of Andrology and Technology of Swine Embryos, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga, São Paulo, Brazil.
authors.forename: André Furugen Cesar
authors.identifier: http://orcid.org/0000-0003-0222-0544
authors.initials: AF
authors.lastname: de Andrade
mesh_terms: D000177:Acrosome / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D015925:Cryopreservation; D005298:Fertility; D005434:Flow Cytometry / Q000379:methods*; D007700:Kinetics; D008297:Male; D051336:Mitochondrial Membranes / Q000378:metabolism; D010766:Phosphorylation; D012661:Semen / Q000166:cytology*; D055101:Semen Analysis; D013094:Spermatozoa / Q000166:cytology*; D034421:Sus scrofa; D014443:Tyrosine / Q000378:metabolism
chemical_list: D014443:Tyrosine