Short communicationTotal antioxidant capacity of fish seminal plasma
Introduction
Oxidative stress (OS) is an important factor in the pathophysiology of semen (Aitken and Krausz, 2001, Bennetts and Aitken, 2005). OS occurs as a result of excessive production of reactive oxygen species (ROS) which overwhelms the antioxidant defense system which can lead to cellular pathology (Agarwal et al., 2005). ROS are continuously produced by cellular aerobic metabolism. The unique structure of spermatozoa makes them vulnerable to OS, mainly due to the low volume of cytoplasm which is a rich source of antioxidative enzymes. In semen, the main sources of ROS are immature spermatozoa and leucocytes (Aitken et al., 1992). OS leads to number of pathological changes, including damage to plasmatic membranes and DNA. Sperm plasma membranes contain lipids in the form of polyunsaturated fatty acids, which are vulnerable to attack by ROS (Wathes et al., 2007). Elevated ROS production is linked to potential damage to sperm DNA, due to its fragmentation (Duru et al., 2000, Said et al., 2005). Another mechanism of damage caused by ROS is oxidative damage to proteins (Tvrda et al., 2011). Low levels of antioxidants are related to low quality of semen (Abd-Elmoaty et al., 2010, Shamsi et al., 2009).
Enzymatic (oxidant defensive enzymes) and non-enzymatic antioxidants are designed to counteract cellular damage caused by excessive production of ROS (Kefer et al., 2009). The main enzymes with the role of oxidant scavengers include superoxide dismutases (SOD), catalase and glutathione peroxidase. Non-enzymatic antioxidants include glutathione and other thiol compounds (Luberda, 2005), albumin and other proteins, ascorbic acid, alpha-tocopherol, carotenoids, uric acid, tyrosine, taurine, hypotaurine (Tvrda et al., 2011). Enzymes are the main part of intracellular defense of spermatozoa against ROS, whereas extracellularly spermatozoa are protected by components of seminal plasma, both enzymatic and non-enzymatic ROS scavangers (Van Overveld et al., 2000).
Spermatozoa of teleost fish are often stored within the reproductive system for a prolonged time, often several months (Ciereszko et al., 2000). During this period, conditions of storage should protect the fertilizing ability of sperm, its motility and maintain metabolism to preserve viability and energetic resources for sperm activation. Therefore, adequate level of antioxidant protection during this period seems to be critical. Among non-enzymatic antioxidants, ascorbic acid is present in fish seminal plasma, despite the inability of teleost fish for its synthesis. Ascorbic acid concentrations in semen are regulated by its dietary levels (Ciereszko and Dabrowski, 1995). Low levels of ascorbic acid in seminal plasma correlate to damage to male germ cells (Ciereszko et al., 1999a). High concentrations of uric acid were also identified in fish seminal plasma (Ciereszko et al., 1999b). Recently, Lahnsteiner and Mansour (2010) and Lahnsteiner et al. (2010) provided comprehensive analysis of antioxidants in fish semen. Uric acid was found to be the main non-enzymatic antioxidant. As far as non-enzymatic antioxidants are concerned besides uric acid and ascorbic acid, glutathione, methionine, and tocopherol were identified as such. Enzymatic antioxidants are also present in fish semen, of them SOD activity predominates.
Evaluation of antioxidant capacity of seminal plasma is often studied from the perspective of individual antioxidants. This approach is time-consuming due to the necessity of several compounds for analysis and fluctuating levels of individual antioxidants (Lahnsteiner and Mansour, 2010). An alternative methodical approach is to measure total antioxidant capacity (TAC). One of commonly used methods is based on the principle that ferryl myoglobin radical oxidizes 2,2'-azinobis-(3-ethyl-benzothiazoline-6-sulphonic acid) (ABTS) to produce a radical cation, ABTS.+, a green soluble chromogen which can be measured spectrophotometrically. Antioxidants in the sample suppress production of this radical cation in a concentration-dependent manner. TAC was found to be a reliable and simple test for the diagnosis and management of male fertility in humans (Mahfouz et al., 2009). Recently, we measured TAC in the seminal plasma of cod (Butts et al., 2011a) and found it useful for discrimination between wild and cultivated fish (Butts et al., 2011b). Further studies revealed that TAC in chinook salmon is two-times higher than in cod (Flannery et al., 2013). This raises question if TAC in fish seminal plasma is species-specific. The objective of this work was to compare TAC in 13 fish species. Additionally, we measured TAC in a 2 kDa filtrate in order to evaluate participation of low molecular weight and high molecular weight antioxidants in fish seminal plasma. Moreover, measurement of TAC in blood plasma was attempted in order to evaluate differences between blood and semen.
Section snippets
Source of semen and blood
Northern pike Esox lucius (L.), Prussian carp Carassius gibelio (Bloch), chub Leuciscus cephalus (L.), carp Cyprinus carpio L. and dace Leuciscus leuciscus (L.) males were caught in a fish pond of the Knieja Fishery Farm near Częstochowa (Southern Poland). Males of barbel Barbus barbus (L.) originated from Czarci Jar near Olsztyn (North-eastern Poland) and were caught in earthen ponds. Asp Aspius aspius (L.) males were caught in the Pierzchały Dam Reservoir located in northern Poland on the
Values TAC and protein concentrations in seminal plasma
Variable TAC values among species were found (Table 1), from 0.008 mM of Trolox (close to detection limit) for rainbow trout to 1.909 mM for Eurasian perch (samples had to be diluted 5–15-times). Protein concentrations did not correlate with TAC within species with exception of Prussian carp (Table 1). An overall plot of individual data is shown in Fig. 1.
Values of TAC in seminal plasma and 2 kDa filtrate
Most of TAC values of seminal plasma (78 – 85%) was recorded in filtrate (Table 2). TAC values of filtrate of perch seminal plasma after second
Discussion
In this study, we have found that TAC values are quite variable among 13 species studied. Two species of percid fish were distinguished by high values of TAC. Most of TAC was attributed to fraction of ≤ 2 kDa. Under conditions of the antioxidant assay kit used in our study, it was impossible to measure TAC in fish blood.
Our results demonstrate remarkable variability in TAC in fish seminal plasma, TAC values in perch was about 200 times higher than in rainbow trout. Therefore, it can be suggested
Acknowledgements
The authors would like to thank the anonymous reviewer for valuable comments and suggestions. This study was supported by funds appropriated to Institute of Animal Reproduction and Food Research and funds from Committee for Scientific and Technological Cooperation between Republic of Hungary and Republic of Poland.
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