Supplementation of soybean lecithin-based semen extender by antioxidants: complementary flowcytometric study on post-thawed ram spermatozoa

Abstract

The purpose of the current study was to evaluate the effects of cysteine (C) and glutathione (G) on the post-thawed ram sperm quality. Collected semen samples from four mature rams were diluted with five soybean lecithin (SL)-based extenders containing: no antioxidant (SL-0), 5 mM cysteine (SL-C5), 10 mM cysteine (SL-C10), 5 mM glutathione (SL-G5) and 10 mM glutathione (SL-G10). After freeze-thawing process, motion and velocity parameters, plasma membrane integrity and functionality, morphological abnormality, lipid peroxidation, acrosomal status, mitochondria activity, and apoptosis status of post-thawed ram spermatozoa were assessed. The results showed that SL-C10 increased the total motility and plasma membrane integrity (p < 0.05) of post-thawed ram spermatozoa (55.86 ± 1.37 and 60.57 ± 1.34 %) compared to other extenders. Progressive motility was significantly higher in SL-C10 (24.71 ± 1.13 %) compared to SL-0 (20 ± 1.13 %) and SL-G10 (15 ± 1.13 %). Mitochondrial activity was significantly higher in SL-C10 (56.83 ± 2.29 %) compared to SL-G10 (38.75 ± 2.29 %). Capacitation and acrosomal status, lipid peroxidation, and the percentage of dead spermatozoa were not affected by different extenders. The percentage of live spermatozoa was higher in SL-C10 (56.33 ± 1.35 %) compared to other extenders. Also, SL-C10 resulted in a lower percentage of apoptotic spermatozoa (14.17 ± 0.53 %) compared to other extenders. The results of this study showed that supplementation of SL-based ram semen extender with 10 mM cysteine resulted in an improved quality of post-thawed ram spermatozoa.

Cysteine protects rabbit spermatozoa against reactive oxygen species-induced damages

“…These data indicate that cysteine protects rabbit spermatozoa from damage during cooling, freeze-thawing, and post-thaw incubation. In the previous studies, addition of cysteine to freezing extender increased the motility and membrane integrity of the frozen-thawed spermatozoa in cats [20], buffalos [14], goats [18, 19], rams [21], and boars [9]. Furthermore, addition of cysteine significantly decreased the level of 8-OHdG, which is a biomarker for DNA oxidative damage.…”

“…Uysal and Bucak (2007) [7] reported that cysteine enhanced intracellular glutathione (GSH) biosynthesis and protected the proteins, DNA and membrane lipids because of the direct radical-scavenging ability of GSH. It has been shown that cysteine protects spermatozoa against cryo-damage in boars [8, 9], bulls [10, 11], buffalos [12–14], dogs [15], chickens [16], goats [17–19], cats [20], and rams [6, 7, 21, 22]. However, the underline mechanisms are still unclear.…”

Fertility and flow cytometry study of frozen-thawed sperm in cryopreservation medium supplemented with soybean lecithin

“…However, possible problems of egg yolk such as bacterial contamination and quality variation have been reported [8,17]. Therefore, it seems that replacement of egg yolk by an alternative cryoprotectant has several advantages, such as improvement of consistency in the semen extenders components, hygienic risks elimination and improvement of fertility after freeze-thawing [31].…”

“…Soybean lecithin (SL) has recently been successfully applied as an extracellular cryoprotectant for cryopreservation of semen in ram [11,30], goat [27], bull [1], buffalo [2] and human [23]. In recent years, SL has been a replacement for egg yolk (EY) due to increment the risk of microbial contamination of egg yolk base extenders [31]. In addition, EY increases the agglutination and pre-capacitation of sperm that has crucially effects on sperm quality [9,16].…”

Current status and advances in ram semen cryopreservation

“…Low-molecular weight amino acids were widely used as additives in ram semen extenders. Among them methionine, cysteamine and cysteine were extensively studied and were indicated as supplements that can reduce malondialdehyde's levels and improve post-thaw ram semen parameters Najafi et al, 2014;Sharafi et al, 2015;Toker et al, 2016). However, there are relative to the aforementioned amino acids studies, which report negative effects of cysteamine on semen motility, mitochondrial activity and DNA integrity (Cirit et al, 2013; Mata-Campuzano et al,…”