Preservation of boar semen at 18 °C induces lipid peroxidation and apoptosis like changes in spermatozoa

Abstract

Boar sperm functions, lipid peroxidation status, mitochondrial membrane potential (Δ Ψ m) and membrane permeability (apoptosis like features) were assessed during liquid preservation. Four ejaculates each from four Hampshire boars were extended with Beltsville Thawing Solution and preserved at 18 °C. At 0, 24, 48, 72 and 96 h of storage, each ejaculate was examined for sperm functions, lipid peroxidation, Δ Ψ m, and membrane permeability. The lipid peroxidation status of the sperm was assessed based on the malonaldehyde (MDA) levels. Detection of Δ Ψ m was done using 3,3′-dihexyloxacarbocyanine iodide [DiOC 6(3)]/propidium iodide (PI) assay and Yo-pro-1/PI assay was used to detect change in plasma membrane permeability. The sperm motility, viability and acrosomal integrity declined significantly ( p < 0.05) from 0 to 96 h of preservation. At the start of the preservation, the MDA levels (nM/10 9 sperm) were low in sperm (99.83 ± 2.69) and seminal plasma (191.98 ± 11.58), which gradually increased up to the 96 h of storage. Highest negative correlation ( r value) was observed between MDA levels and sperm motility (−0.97), live percent (−0.97), acrosomal integrity (−0.97) and hypo-osmotic sperm swelling test (HOSST) positive sperm percentage (−0.98). Strong positive correlation was observed between HOSST positive sperm percentage and intact acrosome percentage ( r = 0.98). There was a significant ( p < 0.05) increase in the sperm cells with low Δ Ψ m from 0 to 96 h of preservation. Before preservation, 14.85 ± 4.66% of sperm cells of the ejaculate showed low mitochondrial membrane potential, whereas after 96 h of preservation, this proposition of cells increased up to 32.00 ± 6.25%. The apoptotic sperm population was 8.33 ± 2.31% in fresh semen, while this population was 25.19 ± 4.25% at 96 h of preservation and the difference was significant ( p < 0.05). The findings of the present study revealed that liquid preservation of boar semen at 18 °C induces lipid peroxidation, decrease mitochondrial membrane potential and increase the plasma membrane permeability.