Roles of lipid peroxidation and cytoplasmic droplets on in vitro fertilization capacity of sperm collected from bovine epididymides stored at 4 and 34 °C

Abstract

Sperm recovery from the cauda epididymis can be very advantageous, for example, in case of the unexpected death of a genetically highly valuable animal, for preserving endangered species, or when the collection of sperm by other means becomes impossible. Studies indicate that epididymides stored at cooler temperatures result in better quality sperm. One of the factors that could negatively affect sperm viability during storage is lipid peroxidation, in which the sperm membrane's ability to resist attacks by reactive oxygen species (ROS) plays an important role. Another factor is the presence of cytoplasmic droplets, which appear in high numbers in epididymal sperm, and are known to influence oxidative stress. The objectives of this study were: to determine whether the post-slaughter storage temperature of the epididymis would effect the sperm membrane's resistance to lipid peroxidation and/or the sperm cell's fertilizing capacity in vitro and to elucidate the role played by the cytoplasmic droplets. Forty-eight testicles with epididymides (24 bulls) were collected following slaughter, and divided into two groups. One testicle from each pair was stored at 4 °C, and the other at 34 °C, for 2 h, after which sperm was collected from the caudae epididymides. Sperm concentration was measured, and an aliquot containing 10 8 sperm was subjected to induced lipid peroxidation with ferrous sulphate and ascorbate (37 °C, 2 h). Subsequently, thiobarbituric acid reactive substances (TBARS), as an index of lipid peroxidation, were measured. A second aliquot of the same sample was used in a routine in vitro fertilization performed in duplicate. Sperm from caudae epididymides stored at 34 °C resulted in lower rates of total blastocyst formation and had a higher percentage of distal droplets, when compared to sperm from epididymides stored at 4 °C (21.2 ± 2.42 and 71.8 ± 4.7% versus 33.5 ± 1.8 and 23.7 ± 4.7%, respectively, P < 0.05). Storage temperature had no effect on TBARS levels. For samples stored at 4 °C, TBARS were negatively correlated with distal droplets ( r = −0.63, P < 0.05) and positively correlated with proximal droplets ( r = 0.42, P < 0.05). In conclusion, our results show that short-term storage of epididymides at 4 °C provided sperm of higher quality and in vitro fertilizing capacity than storage at 34 °C. Although resistance to oxidative stress could not be shown to directly influence these results, distal sperm droplets that appeared in high numbers in the cooled epididymal sperm samples, may have exerted an antioxidant effect. We hypothesize that this protection against ROS is one of the functions of distal sperm droplets in the epididymis.