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

Abstract

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 frozenthawed 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™ Deep Red), three dyes (3-panel; SYBR ® 14, propidium iodide and lectin PNA-Alexa™ 647) or four dyes (4-panel; Hoechst 33342, lectin PNA-Alexa™ 488, propidium iodide and MitoTracker™ 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 deadacrosome 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.

Spermatozoa cooled to 5°C one day after collection from porcine commercial semen doses retain sperm functionality with reduced bacterial load

“…Viability, acrosome integrity, and mitochondrial activity were evaluated using a four‐color panel 26 . Briefly, a 12.5‐μL aliquot of each sample was suspended into 112.5 μL HEPES‐buffered saline (HBS) medium (10 m m glucose, 20 m m HEPES, 137 m m NaCl, 2.5 m m KOH, pH 7.2, 300 mOsm/kg) to a final sperm concentration of 3 × 10 6 spermatozoa/mL.…”

Expanding the use of flow cytometry in semen analysis: The rise of flow spermetry

“…This study underlines the utility of flow cytometry in studying the biology of spermatozoa and its potential to rapidly translate the information gathered into clinical settings. Other recent papers published also demonstrate the utility of flow cytometry in clinical sperm analysis [4], and the study of the biology of the spermatozoa [5, 6].…”

Research Progress on the Microregulatory Mechanisms of Fertilization: A Review

“…The AR is a receptor-mediated cell exocytosis process, in which the ZP receptor binds to ZP3, a ZP glycoprotein, phosphorylates tyrosine protein kinase and activates G protein. Then, cholesterol effluxes, the plasma membrane becomes liquefied, and ZP receptors move within the lipid bilayer membrane, inducing fusion of the sperm plasma membrane with the preacrosomal membrane to form a channel that exposes the nucleus via the anterior acrosomal inner membrane (1). Acrosome vesicles release acrosome proteases and other substances to hydrolyze the ZP around the oocyte and form a in vivo 36: 2002-2013 (2022) channel that allows only one sperm can pass through, further promoting oocyte activation.…”