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?

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Torres, Mariana Andrade ; Diaz, Rommy ; Boguen, Rodrigo ; Massami Kitamura Martins, Simone Maria ; Ravagnani, Gisele Mouro ; Leal, Diego Feitosa ; Oliveira, Melissa de Lima ; Donatelli Muro, Bruno Bracco ; Parra, Beatriz Martins ; Meirelles, Flavio Vieira ; Papa, Frederico Ozanan ; Dell'Aqua, Jr., Jose Antonio ; Alvarenga, Marco Antonio ; Moretti, Anibal de Sant'Anna ; Sepulveda, Nestor ; Cesar de Andrade, Andre Furugen

• Document type: Journal article
• Source: PLoS One; v. 11, n. 8 AUG 16 2016.
• Web of Science Citations: 7

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 (R-2 = 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. (AU)

• FAPESP's process: 13/08070-8 - Effects of seminal plasma on motility characteristics and fertility of cryopreserved boar sperm
• Grantee: Mariana Andrade Torres
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 14/22972-7 - Novel flow cytometer techinique for sperm analysis: validation and practical application
• Grantee: Mariana Andrade Torres
• Support Opportunities: Scholarships abroad - Research Internship - Master's degree
• FAPESP's process: 11/23484-8 - Effects of seminal plasma on inhibition/reversal of capacitation-like changes and fertility of cryopreserved boar sperm
• Grantee: André Furugen Cesar de Andrade
• Support Opportunities: Regular Research Grants