Validation of a new multiparametric protocol to assess viability, acrosome integrity and mitochondrial activity in cooled and frozen thawed boar spermatozoa

Background Motility, morphology, membrane integrity and DNA fragmentation are sperm characteristics routinely used to assess quality of boar spermatozoa. However, the evaluation of individual parameters has intrinsic restrictions in the estimation of potential fertility. Therefore, we aimed to validate a new multiparametric protocol to assess fertility potential through the evaluation of viability, acrosome integrity and mitochondrial activity within the same sperm population for cooled and frozen-thawed boar spermatozoa. Method Three multicolor protocols to assess viability, acrosome integrity and/or mitochondrial activity were compared for agreement containing two dyes (HM-panel; Hoechst 33342, MitoTracker (TM) Deep Red), three dyes (3-panel; SYBR (R) 14, propidium iodide and lectin PNA-Alexa (TM) 647) or four dyes (4-panel; Hoechst 33342, lectin PNA-Alexa (TM) 488, propidium iodide and MitoTracker (TM) Deep Red). Cooled (n = 132) and frozen-thawed (n = 254) samples of boar spermatozoa were assessed by flow cytometry. Results 4-Panel enabled the detection of several sperm subpopulations based on plasma membrane integrity, acrosome status and mitochondrial activity in cooled and frozen-thawed spermatozoa. No significant differences were observed between 3-panel and 4-panel for the percentage of live, live-acrosome intact, and dead-acrosome reacted spermatozoa. However, the percentage of acrosome-intact spermatozoa was significantly higher in cooled samples when stained by 3-panel than 4-panel. Percentages of sperm parameters between protocols were strongly correlated, and agreement analysis demonstrated that both assays resulted in similar values for both sperm sample type. Conclusion Our results indicate that a four-color protocol is a practical, simple and reliable procedure to simultaneously evaluate boar sperm viability, acrosome integrity and mitochondrial activity under clinical conditions.