Influence of BHT inclusion on post-thaw attributes of human semen

Abstract

The aim of this study was to determine the effect of butylated hydroxytoluene (BHT) supplemented cryopreservation medium on sperm parameters during the freeze-thaw process. In addition, sperm lipid peroxidation, DNA damage, and the amount of reactive oxygen species (ROS) were determined. Semen samples were obtained from 75 donors. Fifteen semen samples were used for optimizing BHT concentration and incubation time and 60 samples were used for the final experiments. After the determination of basic parameters, groups of three sample with similar parameters were pooled and processed by Pure Sperm gradient centrifugation. The semen samples were then diluted with normal freezing medium (control) or a medium containing 0.5 mM BHT (test) for 5 minute and stored in liquid nitrogen. Frozen cryovials were thawed individually for 20 seconds in a water bath (37°C) for evaluation. Freezing extenders supplemented with 0.5 mM BHT led to higher sperm motility and viability compared with control samples (p < 0.001). Furthermore, the addition of BHT decreased malondialdehyde (MDA) formation, DNA fragmentation, and ROS content compared with controls (p < 0.001). Our results showed that the addition of BHT to the freezing medium could be of advantage in reducing ROS and preventing the detrimental effect of ROS on the human sperm function.

• Butylated hydroxytoluene
• cryopreservation
• DNA fragmentation
• reactive oxygen species
• spermatozoa

Introduction

Cryopreservation is the procedure that stabilizes the cells at cryogenic temperatures. Maintenance of male and female gametes by cryopreservation technology has progressed in recent years [Gadea et al. 2011]. In spite of the success of sperm cryopreservation, cell damage and defect in sperm function have been reported using this technique. It is shown that freezing and thawing decreases sperm motility and viability but also causes an increase in sperm DNA damage [Thomson et al. 2009; Zribi et al. 2010]. Defects in sperm function as well as cell and membrane injury may be related to cold shock, osmotic stress, and intracellular ice crystal formation during cryopreservation [Watson 2000]. Sperm membrane fluidity and mitochondrial function are damaged by freeze-thawing. It has been hypothesized that freezing and thawing of spermatozoa results in an increased amount of reactive oxygen species (ROS) which could damage cellular compounds including DNA, lipids, and proteins [Thomson et al. 2009; Zribi et al. 2012]. Although a low concentration of ROS promotes maturation and capacitation of spermatozoa, excessive ROS can lead to oxidative stress [Aziz et al. 2010]. Oxidative stress promotes peroxidation of membrane lipids and induces DNA damage [Aziz et al. 2010; Zribi et al. 2012]. In recent years, researchers have tried to diminish the effects of ROS and improve quality of thawed sperms with the addition of antioxidant supplements to the freezing medium. Antioxidants such as ascorbate [Li et al. 2010], vitamin E [Kalthur et al. 2011], resveratrol [Branco et al. 2010], and reduced glutathione [Gadea et al. 2011] have been tested during human sperm cryopreservation.

Butylated hydroxytoluene (BHT), an antioxidant with lipophilic properties, could convert peroxy radicals to hydroperoxides and diminish auto-oxidation. BHT has been tested in animal studies [Ijaz et al. 2009; Khalifa et al. 2008; Memon et al. 2011; Roca et al. 2004; Shoae and Zamiri 2008]. Aitken and Clarkson [1988] showed that the addition of BHT into the media, used in sperm isolation techniques, decreases ROS production during centrifugation of spermatozoa. However, limited information is available on the applicability of BHT supplement as an antioxidant in cryopreservation medium of human spermatozoa. During sperm cryopreservation, reducing the amount of ROS, and thus decreasing lipid peroxidation and DNA damage of sperm cells, is important. We hypothesize that BHT might have beneficial effects on sperm cell membrane and DNA integrity. The main aim of this study was to determine the effect of BHT supplemented cryopreservation medium on sperm parameters, sperm lipid peroxidation, DNA damage, and ROS content during the freeze-thawing process.

Results

Semen samples from 75 donors (age 25–35 years) with normal semen analysis parameters were obtained by masturbation following three days of abstinence, according to World Health Organization [WHO 2010] guidelines. Fifteen semen samples were used for optimizing BHT concentration and incubation time and 60 samples were used for final experiments. Semen parameters of all participants are illustrated in Supplemental Table 1. The study was approved by the Ethical Committee of Hamadan University of Medical Sciences and written consent was obtained from all participants.

Table 1. Determination of optimum dose of butylated hydroxytoluene (BHT) in freezing medium according to percent of motility, viability, anion superoxide (), and hydrogen peroxide (H₂O₂).

	After thawing
Group	Motility (%)	Viability (%)	(%)	H2O2 (%)
Control	35 ± 5	46 ± 10	53 ± 3	68 ± 5
BHT 3 mM/15 min	30 ± 5	45 ± 5	60 ± 5	63 ± 10
BHT 3 mM10 min	30 ± 5	45 ± 10	58 ± 5	67 ± 5
BHT 3 mM/5 min	30 ± 5	46 ± 5	58 ± 5	67 ± 5
BHT 1.5 mM/15 min	34 ± 5	40 ± 5	55 ± 5	65 ± 8
BHT 1.5 mM/10 min	35 ± 5	45 ± 10	55 ± 5	65 ± 5
BHT 1.5 mM/5 min	38 ± 5	45 ± 5	50 ± 5	65 ± 5
BHT 1 mM/15 min	35 ± 5	40 ± 10	50 ± 5	65 ± 3
BHT 1 mM/10 min	38 ± 2	45 ± 3	50 ± 5	58 ± 5
BHT 1 mM/5 min	35 ± 5	45 ± 10	45 ± 10	58 ± 5
BHT 0.5 mM/15 min	40 ± 5	42 ± 5	47 ± 8	56 ± 5
BHT 0.5 mM/10 min	40 ± 5	50 ± 5	45 ± 5	55 ± 5
BHT 0.5 mM/5 min	44 ± 5*	56 ± 10*	43 ± 5*	52 ± 5*

All semen samples were processed with PureSperm gradient and sperm parameters were determined after thawing (n = 5). *p < 0.05

In order to determine the impact of BHT on semen parameters, the optimum concentration of BHT was defined. Then, the defined concentration of BHT was applied to all of the semen samples. Briefly, fifteen semen samples with similar characteristics, including motility grades, viability, morphology, and concentration were randomly divided into five sets of three semen samples. Then, semen samples from each set were pooled (five pooled samples), and used to determine the optimum incubation time and desired dose of BHT.

Optimizing BHT supplementation

To prepare BHT solutions, BHT powder was dissolved in ethanol to different concentrations of BHT that ranged from 3.0, 1.5, 1, and 0.5 mM. Then, one ml of each BHT solution was transferred into a cryotube. The ethanol was then evaporated and a thin crystallized layer of BHT was deposited on the cryovial tubes. In the next step, crystalized BHT was dissolved in 0.5 ml of Quinn’s Advantage Sperm Freezing medium (SAGE In-Vitro Fertilization Inc., Trumbull, CT, USA). In addition, a control cryotube was prepared without BHT. To determine the impact of BHT on semen parameters, each of the different BHT concentrations was applied onto five pooled semen samples. Briefly, aliquots of 1 ml of the liquefied pooled semen samples were layered on top of the upper layer of 40% and 80% PureSperm gradient (Nidacon International, Sweden) and centrifuged at 300 × g for 20 minutes. After removal of the supernatant, the pellets were washed with 5 ml of cook sperm medium (COOK IVF, Australia) and centrifuged at 500 × g for 10 minutes [Amiri et al. 2012]. Then, the pellets were resuspended in 0.5 ml of cook sperm medium. Finally, sperm samples were transferred into a series of cryotubes (5 tubes) containing one of different BHT concentrations (e.g., 0.5 mM) and tubes were incubated for 5, 10, or 15 minutes at 37°C to allow uptake of BHT by spermatozoa. This was then repeated for the other concentrations of BHT. Finally, semen samples were slowly frozen in liquid nitrogen vapors [Ijaz et al. 2009] and stored at −196 °C. After thawing, sperm motility, viability (detected by staining with eosin 0.5%), and ROS content were evaluated. The result for determining the optimum dose and incubation of BHT are described in Table 1. Our findings indicated that the 0.5 mM BHT concentration and a 5 minute incubation time are the optimum conditions for sperm cryopreservation (p < 0.05).

Effect of BHT on post-thaw semen quality

The influence of the optimum BHT concentration and time of incubation of BHT on post-thaw attributes was examined using semen samples from 60 donors with similar parameters. They were pooled, as described above, to obtain 20 pooled samples and the optimum sperm cryopreservation conditions were applied to the pooled samples. After thawing the cryosperm samples were gently mixed and washed with cook sperm medium (COOK IVF, Australia) to eliminate cryosperm media [Ijaz et al. 2009] then sperm motility, viability, DNA fragmentation, malondialdehyde (MDA), and ROS content were evaluated (all experiments were carried out in duplicate).

Data for the effects of BHT supplementation on sperm characteristics are shown in Table 2. Significant differences were observed in motility, viability, MDA, DNA fragmentation, and the concentration of H₂O₂ and (p < 0.001). Motility and viability were significantly higher in the BHT treated group compared with control group (p < 0.001), whereas MDA, amount of H₂O₂, amount of and DNA fragmentation (Figure 1) were significantly lower in the sperm samples that received the BHT antioxidant (p < 0.001).

Figure 1. Determination of DNA fragmentation in the thawed sperm of control group (A), no addition of butylated hydroxytoluene) and in the thawed sperm incubated 5 minutes with 0.5 mM butylated hydroxytoluene (B) using the TUNEL test. Spermatozoa with fragmented DNA were visualized using fluorescence microscope in excitation wavelength 488 nm and detection in the 540 nm. The spermatozoa with DNA fragmentation are shown in green and spermatozoa having intact DNA are shown in red.

Table 2. Level of sperm motility, viability, MDA, amount of H₂O₂, amount of , and DNA fragmentation of frozen sample (n = 20) with or without 0.5 mM butylated hydroxytoluene (BHT).

Group	Motility (%)	Viability (%)	MDA (nm/10 × 10^6)	H2O2 (%)	(%)	Damaged DNA (%)
Control	34.7 ± 5.7	46.56 ± 5.9	0.35 ± 0.03	68.21 ± 2.96	53.3 ± 5.7	49 ± 7.6
BHT 0.5 mM/5 min	44.4 ± 5.9	56.04 ± 5.7	0.32 ± 0.04	52.52 ± 9.16	43.3 ± 4.71	25.3 ± 7.6
p values	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001

The data are reported after thawing of samples.

Discussion

The data reported above shows that sperm preservation with freezing media supplemented with 0.5 mM BHT, resulted in higher sperm motility and viability concurrent with a lower ROS concentration and DNA damage. In other words, BHT concentration greater than 0.5 mM results in lower sperm motility and viability which is probably due to an increase of superoxide anion and H₂O₂ in sperm cells. Our results are consistent with observations made by Shoae and Zamiri [2008] who reported toxicity of BHT at concentrations greater than 1 mM causing increased ROS levels and decreased sperm motility and viability. Other antioxidants including ascorbate, vitamin E, resveratrol, and reduced glutathione have been studied for cryopreservation of human spermatozoa. These studies showed that cryopreservation of human spermatozoa increased oxidative stress and the use of antioxidants can reduce the stress induced oxidative damage in postthaw spermatozoa [Branco et al. 2010; Gadea et al. 2011; Kalthur et al. 2011; Li et al. 2010].

Freezing and thawing stimulates ROS formation which can manifest as lipid peroxidation of the sperm cell membrane. Human spermatozoa cell membranes are rich in polyunsaturated fatty acids, such as docosahexaenoic acid and eicosapentaenoic acid which are highly sensitive to ROS attack and peroxidation [Tavilani et al. 2006; Tavilani 2007]. Peroxidation of polyunsaturated fatty acids in membrane phospholipid has been known to impair the sperm cell and manifest as membrane damage and decrease in motility [Tavilani et al. 2008]. In this study, we investigated the effects of the BHT supplement on lipid peroxidation by determing MDA concentration. Our results showed that the addition of BHT into the freezing medium significantly decreases sperm MDA concentration. Aitken and Clarkson [1988] reported that supplementation of sperm isolation media with BHT effectively inhibits lipid peroxidation of human spermatozoa during repeated centrifugation. Tavilani et al. [2005] have previously reported a relationship between MDA concentration and loss of sperm motility in infertile individuals. The results of the present study suggests that using BHT reduces the level of MDA and consequently improves sperm motility.

Freezing and thawing has deleterious effects on sperm DNA integrity by inducing DNA fragmentation [Thomson et al. 2009; Zribi et al. 2010]. DNA integrity of sperm is necessary for the correct transmission of genetic information to the next offspring and therefore, DNA damage may result in male infertility [Agarwal and Said 2003]. Evidence suggests that high levels of ROS during the freezing and thawing mediate the occurrence of single- and double-strand DNA breakage in the sperm nucleus. Thomson et al. [2009] reported that cryopreservation of human sperm cells stimulated DNA damage which is mediated by oxidative stress. Here, we showed that the addition of BHT into the freezing media reduces the percent of sperms with DNA damage from 49% in non-treated cells to 25% in BHT-supplemented cells. Therefore, it can be concluded that the addition of BHT to the freezing media reduces spermatozoa ROS formation and consequently DNA fragmentation.

Our results showed that during thawing, there is an increased level of ROS (superoxide anion and H₂O₂) in the control sample compared with BHT-treated samples. Lasso et al. [1994] reported that loss of superoxide dismutase due to membrane damage and leakage of this enzyme from the cell result in increasing superoxide anion during cryopreservation. Cryopreservation reduces sperm superoxide dismutase activity by 50% [Bilodeau et al. 2000]. In addition, Gadea et al. [2011] reported that the high level of H₂O₂ during the freeze-thaw process is due to loss of glutathione and reduction of glutathione reductase activity. In the present study, a significant reduction in ROS formation was observed when BHT was added to the freezing medium. This is in agreement with the findings of Aitken and Clarkson [1988] who reported that BHT supplementation into the sperm isolation media results in a decrease in ROS generation by spermatozoa. It seems that BHT could be an important scavenger of free radicals in the spermatozoa freezing medium and can compensate for reduced activity of superoxide dismutase and glutathione peroxidase after a cycle of freezing and thawing. In conclusion, the addition of BHT to the freezing medium could be of advantage in reducing ROS formation and preventing the detrimental effect of ROS to human sperm function.

Materials and Methods

Semen collection

The study was approved by the Ethical Committee of Hamadan University of Medical Sciences and written consent was obtained from all participants. Semen samples from 75 donors with normal semen analysis were obtained by masturbation according to WHO [2010] guidelines. Fifteen semen samples were used for optimizing BHT concentration and incubation time and 60 samples were used for final experiments.

Study design

To determine the impact of BHT on semen parameters, the optimum concentration of BHT and the optimum incubation time were first defined. Briefly, 15 semen samples with similar characteristics were randomly divided into five sets of three semen samples. Then, semen samples from each set were pooled and used in a time-concentration course experiment. After evaluating sperm motility, viability, and ROS content, 5 min incubation of sperms with 0.5 mM BHT concentration was determined as an optimized condition.

Finally, semen samples from 60 donors with similar parameters were pooled, as described earlier, to obtain 20 pooled samples, incubated 5 min with 0.5 mM BHT, and were slowly frozen in liquid nitrogen vapors. After thawing of frozen cryosperm, samples gently were mixed and washed with cook sperm medium to eliminate cryosperm [Ijaz et al. 2009] and sperm motility, viability, DNA fragmentation, MDA, and ROS content were evaluated.

Viability test

The viability of the sperm in the sample was assessed by means of eosin 0.5% stain. The sperm smears were prepared by mixing a drop of semen with one drop of the stain on a warm slide. The viability was assessed by counting 200 cells under 400 × magnification. Sperms with partial or complete red colorization were assigned non-viable or dead whereas colorless sperms were designated as alive [WHO 2010].

Lipid peroxidation

Lipid peroxidation in spermatozoa was measured as TBARS (Thiobarbiuric Acid Reactive substances) procedure, according to Yagi’s methods [Yagi 1984]. MDA concentration was measured spectrofluorometrically using a Jasco FP-6200 spectrofluorometer (JASCO, Japan, excitation 515 nm, emission 553 nm). The MDA fluorescence intensity of spermatozoa was determined using various concentrations of tetraethoxypropane as standards. The results are expressed as nmol of MDA/10 × 10⁶ cells.

Evaluation of sperm DNA fragmentation

Determination of sperm DNA fragmentation was carried out using Cell Death Detection Kit (Roche Diagnostics, Deutschland GmbH, Germany). Thawed sperms were smeared on microscope slides, air-dried, fixed with 4% paraformaldehyde in phosphate-buffered saline for 60 min and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. Then, smears of sperm were further processed for TUNEL assay. Spermatozoa with fragmented DNA were visualized using fluorescence microscope in 488 nm. A negative control (sperm without the addition of the Tdt enzyme) and a positive control (sperm treated with a 141 µM solution of H₂O₂) were also assessed by TUNEL assay [Greco et al. 2005].

Flow cytometric determination of ROS

DCFH-diacetate (for detection of H₂O₂) and Hydroethidium (HE, for detection of ) purchased from Sigma-Aldrich (Chemie Gmbh Munich, Germany) were added to the sperm suspension. Samples were then incubated at 25°C for 40 min for DCFH and for 20 min for HE as previously described by Mahfouz et al. [2009]. A Partec flow cytometer (Munster, Germany) equipped with a 488-nm argon laser as a light source was used for determination of ROS. DCFH-diacetate was evaluated at 500–530 nm (green fluorescence) and HE was evaluated at 590–700 nm (red fluorescence). Unviable spermatozoa were excluded using propidium iodide and Yo-Pro-1 as counter stain dyes for DCFH and HE, respectively [Mahfouz et al. 2009]. Results were expressed as the percentage of fluorescent spermatozoa.

Statistical analysis

Statistical analysis was performed using SPSS 13 (SPSS Inc, Chicago, IL, USA) and results were expressed as the mean ± SD. For determining BHT optimum dose andincubation time (experiment 1), we used repeated measures ANOVA and Tuckey’s post hoc test. To assess the normality of the evaluated variables, Kolmogorov Smirnov test was performed. We used t-test to detect differences between two groups. p < 0.05 was considered as statistically significant.

Abbreviations
BHT	=	butylated hydroxytoluene
ROS	=	reactive oxygen species
MDA	=	malondialdehyde

Author contributions

Designed the study, wrote the paper, and contributed in the critical revision: HT, IK, IA; Contributed in sample collection and performing experiments: MG, MA, AF.

Acknowledgement

This research was financially supported by Hamadan University of Medical Sciences.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. The authors are academic members or postgraduate students of Hamadan University of Medical Sciences. Other authors are postgraduate students in Clinical Biochemistry.