Membrane stability and mitochondrial activity of bovine sperm frozen with low-density lipoproteins and trehalose.

Abstract

Cryopreservation results in the destabilization of the sperm plasma membrane, leading to negative side effects such as premature cryocapacitation, apoptosis and the low mitochondrial activity of bovine spermatozoa. Low-density lipoproteins (LDL) and trehalose have been used in seminal freezing to protect the integrity and stability of sperm membranes. Likewise, trehalose can increase the mitochondrial activity of sperm. The objective of this study was to evaluate the membrane stability and mitochondrial activity of bovine sperm after being frozen and treated with LDL sources and trehalose. Ten ejaculates from five bulls were cryopreserved under the treatments, CEY: chicken egg yolk (20% v/v); CCEY: centrifuged CEY (20% v/v); LDL: LDL (8% v/v); T: trehalose (100mM); and TLDL: T (100mM) plus LDL (8% v/v). After thawing, membrane stability and mitochondrial membrane potential (ΔΨM) were assessed by flow cytometry through the M-540/Yopro-1 and DiOC6/PI probes. The structural membrane integrity (SMI) was evaluated by fluorescence microscopy using SYBR14/PI dyes. A generalized linear model was adjusted, and the means were compared using the Tukey test. Centrifuged chicken egg yolk and LDL had a higher proportion of non-cryocapacitated non-apoptotic sperm (M-Y-), while CEY and T had the largest populations of cryocapacitated non-apoptotic sperm (M+Y-) and cryocapacitated apoptotic sperm (M+Y+). Centrifuged chicken egg yolk also showed a higher proportion of sperm with high-ΔΨM. Treatments that included egg yolk or purified LDL had a positive effect on SMI. Centrifuged chicken egg yolk has a superior cryoprotective effect on membrane stability and mitochondrial activity of bovine semen over the conventional use of CEY or the individual or simultaneous use of LDL and trehalose.