Original ResearchUsing Resveratrol and Epigallocatechin-3-Gallate to Improve Cryopreservation of Stallion Spermatozoa With Low Quality
Introduction
Equine semen cryopreservation is an important method to protect their genetic resources. Despite the benefits of frozen-thawed semen, cryopreservation is understood to cause cryodamages (lethal or sublethal) to the spermatozoa [1], due in part to the increased formation of reactive oxygen species (ROS), and therefore, oxidative damage, which reduces the fertilizing capability of sperm used for artificial insemination (AI) [2]. Oxidative stress is one of the major causes of defective sperm function due to the fact they contain high concentrations of polyunsaturated fatty acids [3], [4] and have limited repair mechanisms [5].
The cytoplasmic origin is the primarily protecting antioxidant system in spermatozoa. Spermatozoa discard most of their cytoplasm during the terminal stages of differentiation, as a consequence lack the significant cytoplasmic component containing antioxidants that counteract the damaging effects of ROS and lipid peroxidation (LPO). Correspondingly, spermatozoa are susceptible to LPO throughout cryopreservation and thawing, leading to subsequent sperm dysfunctions [3], [6]. Generation of ROS is significantly increased in the presence of cryodamaged, nonviable, or morphologically abnormal sperm, particularly sperm characterized by the presence of proximal cytoplasmic droplets or abnormalities of the midpiece [7], [8].
There are a number of antioxidant systems in spermatozoa and seminal plasma that scavenge ROS and prevent internal cellular damage [9], [10]. ROS scavengers are present in seminal plasma, with the primary ROS scavengers described in equine semen being glutathione peroxidase, superoxide dismutase, and catalase. In equine, sperm centrifugation, used to remove seminal plasma and concentrate spermatozoa before freezing, removes antioxidants present in semen, thus exposing spermatozoa to excessive ROS damage [11]. One way to improve sperm viability, and so fertilizing capacity, would be the addition of antioxidants to the freezing medium [12], [13]. Various attempts have been made to minimize/avoid oxidative stress in sperm by alteration of environmental conditions or the use of antioxidants [14], [15], [16], [17], especially during sperm freezing and thawing [16], [18]. In spite of the benefits of antioxidant therapy [18], results vary according to animal species, extender, and the type and concentration of antioxidant used [19].
Resveratrol (3, 40, 5-trihydroxy-trans-stilbene) (Res) is a polyphenolic natural product with a stilbene structure isolated at first from the roots of white hellebore in 1940 [20] and later from Polygonum cuspidatum, a medicinal plant. Today, it is widely consumed in the Mediterranean diet in the form of peanuts, grapes, and wine. Resveratrol shows many biological activities such as antiinflammatory, cardioprotective, chemopreventive, and antiapoptotic activities [21]. Moreover, Res has been reported to possibly act as an antioxidant; thanks to its ability to reduce mitochondria ROS production, scavenge superoxide radicals, inhibit LPO, and regulate the expression of antioxidant cofactors and enzymes [22]. Res is a nonflavonoid polyphenol with intensive antioxidant activity [23].
Juan et al [24] reported that Res can diminish germ cell apoptosis in mice and rats, and increase blood testosterone levels, testicular sperm count, and epididymis sperm motility in rabbits. Phenolic compounds act as free radical scavengers and, on occasion, as metal chelates, acting both in the initiation step, and inducing the oxidative process [11], [12].
Epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea (C. sinensis) and is reported to possess a high level of antioxidant activity [25], [26]. The supplementation of canine sperm with green tea polyphenol extracts (PFs) has been demonstrated to improve motility and viability of spermatozoa during long-term liquid storage [27]. Moreover, pretreatment of boar spermatozoa with PFs before freezing exhibited significantly higher degrees of postthaw sperm viability and acrosomal integrity [28]. The beneficial effect of EGCG has been observed during liquid storage at 15°C of sorted boar semen: it increased the percentage of viable spermatozoa and inhibited caspase activation [29]. Recently, green tea phytocomplexes or polyphenols did not improve stallion semen parameters during cooling at 4°C.
A recent study revealed that the ROS levels are higher in 25% to approximately 40% in semen with low quality [30], [31]. The protective nature of polyphenols and the need to improve the quality of cryopreserved sperm gave rise to the objective of the present study, to assess the effects of the antioxidants Res and EGCG addition to Arabian stallion with poor-quality semen before the cryopreservation process. Also an experiment was designed to assess fertility or subfertility and semen characteristics of our stallions during two breeding seasons. This may prompt the advancement of strategies that facilitate the cryopreservation of important stallions with low-quality semen.
Section snippets
Materials and Methods
Experimental procedures were approved by the University of Zabol Animal Welfare and Ethics Committee.
Unless otherwise noted, all reagents were purchased from Sigma Chemical (St. Louis, MO).
Statistical Analysis
Semen parameters were compared among different treatments groups, analyzed by one-way ANOVA, and expressed as means ± SEM. Percentile data were normalized through arcsine transformation, and then data were submitted to homogeneity testing, followed by variance analysis (one-way ANOVA), with post hoc least significant difference test by IBM SPSS version 19 Statistics Program software (SPSS Inc. Chicago, IL). For all analyses, a P value of <.05 was considered significantly different.
Experiment 1
A total of 81 ejaculates (in two breeding seasons) were collected and examined and semen characteristics are described in Table 1. Evaluation of several ejaculates revealed that sperm production and semen quality were mostly unchanged during the period of evaluation, sperm production was normal, and semen quality was poor. Sperm production was considered normal as sperm output was usually greater than the predicted output based on testicular volume, that is, 6.1 × 109 sperm [32]. The low
Discussion
Abnormal sperms produce more ROS than normal ones; also, ROS levels in semen with low quality are higher in 25% to approximately 40%. In an attempt to reduce and/or minimize the detrimental effects of the oxidative stress, the impact of different concentrations of antioxidants (Res and EGCG) supplementation in the cryopreservation extender of frozen-thawed Arabian stallion semen was investigated. Cold and heat shock during the sperm freeze-thaw process enhances LPO due to ROS production, which
Conclusion
On the basis of the present study, we concluded that Res and EGCG can be good scavengers of free radicals in the spermatozoa freezing medium and can ameliorate sperm motility, viability, membrane integrity, mitochondrial activity, and reduced DNA fragmentation in our stallions with low-quality sperm. The results could be possibly important as the addition of one of these two antioxidants in commercial freezing extenders might enhance sperm fertilizing ability and reproductive performance during
Acknowledgments
This project was financially supported by Mr Seyed Mohammad Mehdi Atashi, the owner of Arabian horse breeding farm in Meybod, Yazd, Iran (Meybod stud). Thus, the authors thank for the facilities provided. This article was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2015R1D1A1A09057348). And this article was written as part of Konkuk University's research support program for its faculty
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Epigallocatechin-3-gallate (EGCG) reduces apoptosis of preantral follicles through the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway after in vitro culture of sheep ovarian tissue
2020, TheriogenologyCitation Excerpt :In our study, EGCG at 1 μg/mL also reduced follicular apoptosis after in vitro culture (Fig. 2). Previously, some studies reported an antiapoptotic activity of the EGCG in bovine oocytes after IVM [12], in equine cryopreserved spermatozoa [26] and in other cells [neuronal: [14]; cardiac: [27]; smooth muscle: [28]; thymocytes: [29]]. Considering that EGCG can penetrate the plasma membrane by passive diffusion [30], we believe that EGCG can act directly on cellular components including nuclei and organelles, and thus enhancing the follicle survival by the inhibition of oxidative stress and prevention of apoptosis [14,29].
Conflict of interest statement: It is declared that all the study was carried out by authors, and there is no conflict of interest.
Animal welfare/ethical statement: Experimental procedures were approved by the University of Zabol Animal Welfare and Ethics Committee.