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ISSN : 2508-755X(Print)
ISSN : 2288-0178(Online)
Journal of Embryo Transfer Vol.32 No.4 pp.337-342

Effects of Antioxidants Supplement in Porcine Sperm Freezing on in vitro Fertilization and the Glutathione and Reactive Oxygen Species Level of Presumptive Zygotes

Sang-Hyoun Park, Yubyoel Jeon, Il-Jeoung Yu†
Department of Theriogenology and Reproductive Biotechnology, College of Veterinary Medicine and Bio-safety Research Institute, Chonbuk National University, Iksan 54596, Republic of Korea
Correspondence: Il-Jeoung Yu +82-63-850-0948,
20171129 20171215 20171226


The present study was aimed to determine the effects of green tea extract (GTE) and beta-mercaptoethanol (β-ME) supplementation in boar sperm freezing extender on in vitro fertilization (IVF) and reactive oxygen species (ROS) and glutathione (GSH) levels of presumptive zygotes (PZs). Experimental groups were allocated into lactose egg yolk (LEY) without antioxidant (control), GTE (1,000 mg/l in LEY) and β-ME (50 μM in LEY). In freezing, spermatozoa extended with LEY were cooled to 5°C for 3 h and then kept at 5°C for 30 min following dilution with LEY containing 9% glycerol and 1.5% Equex STM. The final sperm concentration was 1 × 108/ml. Spermatozoa were loaded into straws and frozen in nitrogen vapor for 20 min. For IVF, oocytes were matured in NCSU-23 medium and co-cultured with spermatozoa following thawing at 37°C for 25 sec. At 12 h following IVF, IVF parameters (sperm penetration and monospermy) were evaluated. In addition, GSH and ROS levels of PZs were determined by Cell Tracker Blue CMF2HC and DCHFDA, respectively. IVF parameters did not show any significant difference among the experimental groups. GSH and ROS levels of PZs were not significantly different between groups. In conclusion, antioxidant supplementation in boar sperm freezing could not influence IVF parameters, ROS and GSH levels of PZs.



    Oxidative stress has been considered as one of the significant causes to reduce male fertility. Sperm cryopreservation is related to an oxidative stress induced by reactive oxygen species (ROS). ROS generation is harmful to the sperm membrane (Watson, 1995) and reduces the sperm parameters including motility, viability and DNA integrity (de Lamirande and Gagnon, 1992; Hu et al., 2008; Waterhouse et al., 2010). Moreover, excessive production of ROS is negatively linked to the sperm function and morphology (Agarwal et al., 2003; Makker et al., 2009; Oborna et al., 2010).

    Various antioxidants, such as α-tocopherol (Olson and Seidel, 2000; Dalvit et al., 2005), L-ascorbic acid (Vitamin C: Tatemoto et al., 2004), glutathione (GSH: Luvoni et al., 1996) and superoxide dismutase (Tatemoto et al., 2004) have been used as ROS scavengers in various species. Yamaguchi et al. (2012) reported that beta-mercaptoethanol (β-ME) supplementation in the sperm thawing solution protect sperm against the lipid peroxidation related with ROS. Green tea (GT: Camellia sinensis) includes various types of catechins inducing antioxidant activity (Dufresne and Farnworth, 2001). Recently, Park and Yu (2017) have demonstrated that the GT extract supplementation in boar sperm freezing extender significantly reduced sperm ROS level after cryopreservation. In other hand, the sperm cells challenged with high concentrations of ROS have many negative biological effects like blocked sperm-egg fusion (Mammoto et al., 1996). However, there is limited information that spermatozoa affected by antioxidants during freezing could affect the oocytes during in vitro fertilization. Therefore, we determined whether the spermatozoa exposed in antioxidants during freezing could play a role in in vitro fertilization (IVF).

    The objectives of the present study were aimed to determine the effects of GTE and β-ME supplementation in boar sperm freezing extender on IVF parameters and ROS and GSH levels of presumptive zygotes (PZs).



    Unless indicated otherwise, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and solutions were prepared with high purity water (6114VF, Sartorius AG, Goettingen, Germany). Equex STM paste was obtained from Nova Chemical Sales Inc. (Scituate, MA, USA).

    2.Preparation of antioxidants

    Green tea powder (GTP) was obtained from Bioland (GTP- 020968, Seoul, Korea). GTE and the β-ME stock were prepared by modifying the stock prepared by Park and Yu (2017). The stock solution of GTE (100,000 mg/l) was prepared by solving GTP in triple distilled water and filtered by 1.2 μm (17593-k, Sartorius, Goettingen, Germany). GTE stock (100,000 mg/l) or β-ME stock (5 mM) was supplemented into the freezing extender (1 : 100 [v/v]). The final concentration of GTE and β-ME in the freezing extender were 1,000 mg/l and 50 μM, respectively.

    High-performance liquid chromatography analysis of 100,000 mg/l GTE revealed 45.9 mg/l catechin, 141 mg/l epicatechin, 297.4 mg/l gallocatechin, 424.3 mg/l epigallocatechin, 392.7 mg/l gallocatechin gallate and 392.7 mg/l epigallocatechin gallate. Chromatography (6430 LC/MS/MS, Agilent, City, USA) was performed at the Center for University-wide Research facilities in Chonbuk National University, Republic of Korea (Park and Yu, 2013).

    3.Collection of semen

    Eight boars (2 Duroc jersey and 6 Yorkshire) were used for this experiment. The semen collected using gloved-hand technique was filtered through four layers of sterile gauze to remove the gel particle. The sperm-rich fraction of ejaculates with greater than 75% motile and 80% morphologically normal spermatozoa were used in this study. The collected semen was pooled to reduce individual differences. The pooled semen were extended (1 : 1[v : v]) in Beltsville thawing solution (BTS; 37 mg/ml glucose, 1.25 mg/ml EDTA, 6 mg/ml sodium citrate, 1.25 mg/ml sodium bicarbonate, 0.75 mg/ml potassium chloride, 0.6 mg/ml penicillin and 1 mg/ml streptomycin) (Pursel and Johnson, 1975). The diluted spermatozoa were assessed for volume, sperm concentration and percentage of motile spermatozoa. Immediately after evaluation, the diluted spermatozoa were stored at 24℃ for 2 h.

    4.Sperm freezing-thawing protocol

    The freezing extenders used in the experiments were composed of freezing extender 1 (FE 1) and freezing extender 2 (FE 2). FE 1 was composed of lactose egg yolk (LEY) extender (80 ml of lactose solution, 20 ml of egg yolk and 0.1% antibiotic-antimycotic in 100 ml sterile non-pyrogenic water). FE 2 consisted of LEY extender supplemented with 9% (v : v) glycerol and 1.5% (v : v) Equex STM. Semen was processed according to the freezing procedure (Guthrie and Welch, 2006). Briefly, semen diluted in BTS was centrifuged at 850 × g for 15 min at room temperature. Following removing supernatant, sperm pellet was resuspended with FE 1 to a concentration of 1.5 × 108/ml. Sperm suspensions were cooled gradually from 24°C to 5°C for 3 h. The spermatozoa were maintained at 5°C for additional 30 min after a second dilution step to 1 × 108/ml with FE 2 at 5°C. The cooled spermatozoa were loaded into 0.5 ml straws and sealed.

    Ten minutes before liquid nitrogen (LN2) vapor freezing, a styrofoam box (29.5 × 18.7 × 24 cm3) was filled with LN2 to a depth of 5 cm and a rack with two bars was set to 7 cm from the surface of the LN2. The straws were then aligned horizontally for 20 min on the rack in the LN2 vapor and then plunged into LN2 for storage. The cooling rates were 30.6°C/min from 5°C to -15°C, 88.2°C/min from -15 to -60.0°C and 14.7°C/min from -60°C to -116.0°C.

    Straws from each group were thawed by immersion in a circulating water bath at 37°C for 25 sec before use in experiments.

    5.Oocyte collection and In vitro maturation (IVM)

    Porcine ovaries were collected from a local abattoir and transported to the laboratory at 34 to 36℃ in 0.9% saline supplemented with 100 IU/ml penicillin G and 100 μg/ml streptomycin. Cumulus-oocyte-complexes (COCs) were aspirated through an 18-gauge needle. Oocytes with compact cumulus mass and a dark, homogenous cytoplasm were washed three times in Tyrode’s lactate-HEPES-polyvinyl alcohol (TL HEPES-PVA: 114 mM NaCl, 3.2 mM KCl, 0.4 mM Na2H2PO4, 2 mM CaCl2ꞏ2H2O, 0.5 mM MgCl2ꞏ6H2O, 5 mM NaHCO3, 20 mM HEPES, 16.6 mM sodium lactate (60% syrup), 0.5% PVA, 10 IU/ml penicillin and 10 μg/ml streptomycin). COCs were cultured in NCSU-23 medium supplemented with 10% porcine follicular fluid (PFF), 0.6 mM cysteine, 10 ng/ml epidermal growth factor, 10 IU/ml pregnant mare serum gonadotropin and 10 IU/ml human chorionic gonadotropin for 22 h and then for another 22 h in maturation medium without hormones at 39℃ in a humidified atmosphere of 5% CO2 in air. PFF was collected from ovarian follicles 3-6 mm in diameter by centrifugation at 1,600 x g for 30 min and filtration through a 1.2 μm syringe filter, aliquoted and stored at - 80℃ until use.

    6.In vitro fertilization (IVF) and In vitro culture (IVC)

    Percoll solutions and gradients were prepared as follows. A two-layer discontinuous gradient was formed by layering 1 ml of a 45% Percoll solution on top of 1 ml of a 90% Percoll solution in a 15 ml conical tube. Aliquants of extended semen (3 ml) were layered onto Percoll gradient and centrifuged for 20 min at 850 × g at room temperature. The pellet recovered after aspiration of the supernatant was washed twice by centrifugation at 350 × g for 3 min with 5 ml D-PBS supplemented with 0.1% BSA, 10 IU/ml penicillin and 10 μ g/ml streptomycin. After the supernatant was discarded, motile spermatozoa were collected. The sperm concentration (spermatozoa/ml) was diluted to 10 × 105 with Tris-buffered medium (mTBM: 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2ꞏ2H2O, 20 mM Tris, 11 mM D-glucose, 5 mM sodium pyruvate, 2 mM caffeine and 0.2% BSA).

    For IVF, the COCs after IVM were denuded by gently pipetting with 0.1% hyaluronidase. Denuded oocytes were washed three times with mTBM and transferred to a mTBM insemination drop (45 μl). A 5 μl volume of spermatozoa was added to each insemination drop to give a final concentration of 1 × 105 spermatozoa/ml. Oocytes and spermatozoa were co-cultured for 6 h at 39℃ in a humidified atmosphere of 5% CO2 in air. Following IVF, presumptive zygotes (PZs) were washed three times, transferred to IVC medium (D-glucose free NCSU-23 supplemented with 0.17 mM sodium pyruvate, 2.73 mM sodium lactate and 0.4% BSA) and incubated for 12 h at 39℃ in a humidified atmosphere of 5% CO2 in the air.

    7.Assessment of sperm penetration and presence of two pronuclei (PN)

    Twelve hours after insemination, the percentages of spermatozoa penetrating oocytes and monospermy were assessed as previously described (Gil et al., 2007). Briefly, the presumptive zygotes were mounted on slides, fixed for 7 days with acetic acid in ethanol (1:3, v:v) at room temperature, stained with aceto-orcein [1% (w:v) orcein in 45% (v:v) acetic acid] and destained with glycerol:acetic acid:DW (1:1:3, v:v) and examined under a light microscope at ×400 magnification. The fertilization parameters evaluated were: penetration (percentage of the number of penetrated oocytes/total inseminated) and monospermy (percentage of the number of monospermic oocytes/total penetrated).

    8.Measurement of intracellular ROS and GSH levels of PZs

    Cell Tracker Blue CMF2HC (4-chloromethyl-6.8-difluoro-7- hydroxycoumarin, Invitrogen) was used to detect GSH levels in PZs as a blue fluorescence. At 12 h after insemination, PZs were incubated for 30 min in TL-HEPES-PVA supplemented with 10 μM Cell Tracker. PZs were washed in TL-HEPESPVA and placed on a glass slide with a 10 μl drop of TLHEPES- PVA.

    The levels of ROS in the PZs were measured in green fluorescence by dichlorodihydrofluorescein diacetate (DCHFDA). After IVC, PZs were transferred into IVC medium containing 10 μM DCHFDA. After 30 min of culture, PZs were washed in DPBS-PVA. The PZs were then placed on a glass slide with a 10 μl drop of DPBS-PVA.

    The fluorescent emissions from the PZs were recorded as TIFF files using a cooled CCD camera attached to a fluorescence microscope (Axio-observer A1, Charl Zeiss, Goettingen, Germany) with filters (excitation: 450-490 nm, emission: 515-565 nm for ROS, UV filter: 370 nm for GSH). The recorded fluorescent images were analyzed using ImageJ software 1.33u (National Institutes of Health, Bethesda, MD, USA) by the intensity of fluorescence in each PZ picture.

    9.Statistical analyses

    Five replicates were conducted for each experiment. Percentage data were subjected to arcsin transformation before analysis. All data are presented as means ± SE and were analyzed using ANOVA followed by Duncan’s multiple range test. Statistical Analysis System ver. 8x software (SAS, Cary, NC, USA) was used. P < 0.05 was considered statistically significant.


    IVF parameters including sperm penetration and monospermy did not show any significant difference among the groups (Table 1).

    The intracellular GSH and ROS levels of PZs are shown in Table 2. Likewise, as the results of IVF parameters, GSH and ROS levels of PZs following IVF with boar spermatozoa cryopreserved with GTE or β-ME or without GTE and β-ME had no significant difference among the groups. However, GTE group showed slightly lower ROS level.Fig. 1

    The increased ROS level causes poor motility, viability, morphology and acrosome integrity of frozen-thawed spermatozoa (Chatterjee and Gagnon, 2001). Bailey et al. (2000) reported that cryoinjury may be induced by ROS activity generated during the process of freezing. Therefore, most researchers have studied the effect of antioxidants during the freezing process for reduction of ROS, (Yamaguchi and Funahashi, 2012). In other word, Gadea et al. (1998) and Sellés et al. (2003) reported that IVF is the most precise tool for evaluating both the refrigerated and frozen-thawed boar semen. Spermatozoa exposed with higher ROS levels could affect on sperm-egg fusion (Mammoto et al., 1996). The reduced ROS level by GTE in frozen-thawed boar spermatozoa was demonstrated (Park and Yu, 2017). Though, the reduced ROS level of frozen-thawed spermatozoa did not make any significant change in IVF parameters of presumptive zygote. Therefore, in the present study, we suggest that the sperm penetration following IVF were not significantly affected by reduced sperm ROS regardless of antioxidant. The effect of antioxidant supplementation during freezing might have limited influence on sperm function in IVF process (giving to porcine oocytes). Similarly, Gadea et al. (2004) reported that supplementation of GSH in sperm freezing extender had no effect on sperm fertilizing ability.

    GSH level is an important indicator of embryo development (Abeydeera et al.,1998; de Matos and Furnus, 2000). GSH supplementation in oocyte maturation medium showed a positive effect on male pronuclear formation during fertilization (Yoshida, 1993; Funahashi et al., 1994). Recent studies also reported that treatment of oocytes with antioxidants during IVM increased embryonic development by reducing ROS and increasing GSH synthesis (Maya-Soriano et al., 2013). Therefore, we inferred that the use of antioxidant in oocyte maturation can induce strong antioxidative effect in the embryo compared to antioxidant used in sperm freezing. In other words, the greater amount of cytoplasm in oocyte than that of sperm head might be more affected by antioxidant. Therefore, use of an antioxidant in sperm freezing extender might not affect embryo development compared to the treatment of antioxidant in oocyte maturation.

    In contrast, addition of GSH to the freezing extender would result in a high rate of ROS-induced GSH oxidation during thawing (Gadea et al 2004). Therefore, we will evaluate the effect of addition of GTE and β-ME in post-thawed spermatozoa to know the latent antioxidant effect on IVF parameters and ROS and GSH level of presumptive zygote.

    On the other hand, we should not overlook the generation of ROS during IVF. Recently the addition of antioxidants to IVF medium exclusively increased mouse blastocyst cell numbers (Truong and Gardner, 2017). The ROS of PZs might be generated from the IVF environment. Therefore, ROS level reduced in only sperm freezing could not influence the ROS and GSH levels of PZs.

    It is concluded that there is no positive effect of supplementation of GTE and β-ME in freezing extender on IVF parameters and ROS and GSH levels of PZs.



    Representative images of the intracellular glutathione (A: GSH) and reactive oxygen species (B: ROS) levels of porcine presumptive zygotes by staining with Cell Tracker Blue CMF2HC and DCHFDA, respectively. A-1 and B-1: control, A-2 and B-2: GTE, A-3 and B-3: β-ME.


    Effects of antioxidants supplementation in sperm freezing extender on fertility parameters

    GTE: green tea extract, β-ME: beta-mercaptoethanol
    The data represent mean ± SE.

    Effects of antioxidants supplementation in sperm freezing on GSH and ROS levels in presumptive zygotes

    GTE: green tea extract, β-ME: beta-mercaptoethanol, ROS: reactive oxidative species
    The data represent mean ± SE.


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