Effects of kojic acid on boar sperm quality and anti-bacterial activity during liquid preservation at 17 C

Theriogenology 140 (2019) 124e135

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Effects of kojic acid on boar sperm quality and anti-bacterial activity during liquid preservation at 17 C Weike Shaoyong, Qian Li, Zhiqiang Ren, Junying Xiao, Zhaoxi Diao, Gongshe Yang, Weijun Pang* Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 March 2019 Received in revised form 10 August 2019 Accepted 20 August 2019 Available online 21 August 2019

Bacteriospermia is a documented risk to sperm quality when boar semen is stored at 17
C. The objective of this study was to evaluate the effects of kojic acid (KA) on sperm quality and anti-bacterial effect during liquid storage boar semen at 17
C, as well as to explore spermeoocyte binding and embryonic development in vitro. Boar semen was diluted with Beltsville thawing solution (BTS), and it contained KA at different concentrations (0, 0.02, 0.04, 0.06, 0.08, and 0.10 g/L). Bacterial concentrations and sperm quality parameters (motility, mitochondrial membrane potential, acrosome integrity, and plasma membrane integrity) were evaluated on each experimental day. Differences in microbial compositions were compared using 16S rDNA sequencing among the control group, 0.04 g/L KA, and 0.25 g/L gentamycin groups on experimental day 5, and the effects of KA on sperm capacitation, Western blot, total anti-oxidant capacity (T-AOC), reactive oxygen species (ROS) content, malondialdehyde (MDA) content, in vitro fertilization (IVF) parameters, spermeoocyte binding, cleavage rates, and blastocyst rates were evaluated. The results showed that KA at the optimum concentration of 0.04 g/L significantly improved sperm quality parameters and sperm capacitation, increased T-AOC ability, enhanced IVF parameters and spermeoocyte binding, increased cleavage and blastocyst rates, inhibited bacterial concentrations, reduced ROS and MDA content, and altered bacterial compositions (P < 0.05). Moreover, KA also increased the expression of anti-oxidant-related proteins, SOD1, SOD2 and CAT, and anti-apoptosisrelated protein, Bcl 2, and decreased the expression of apoptosis-related proteins, caspase 3 and Bax in sperm (P < 0.05). These findings demonstrated that supplementation of antibiotic-free extenders for boar semen with 0.04 g/L KA has beneficial effects on liquid boar sperm preservation. © 2019 Published by Elsevier Inc.

Keywords: Kojic acid Liquid storage Sperm quality parameters Bacterial concentration Embryonic development

1. Introduction In the modern swine production industry, successful artificial insemination (AI) with boar semen subjected to liquid storage at 17
C is commonly used to facilitate pig breeding. Sperm quality is positively correlated with field fertility and is therefore critical for swine AI outcomes. Currently, two main factors, namely, oxidative stress and bacterial contamination, affect sperm quality parameters and fertilization rates [1e4]. Semen collection and preservation are nonsterile procedures that can result in bacterial contamination of boar semen. Therefore, to control the bacterial load during liquid

* Corresponding author. No. 22 Xinong Road, Yangling, Shaanxi Province, 712100, China. E-mail address: [email protected] (W. Pang). https://doi.org/10.1016/j.theriogenology.2019.08.020 0093-691X/© 2019 Published by Elsevier Inc.

storage, antibiotic substances are usually added to boar semen [5]. However, a negative effect of using antibiotics is that most of the bacteria isolated from animal samples develop resistance to common antibiotics [6]. Thus, to reduce the use of antibiotics, it is necessary to identify appropriate replacements for antibiotics. Kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one; KA) is an organic acid which is produced by several species of fungi and bacteria. KA and its derivatives have been reported to have various biological activities. The main applications of KA and its derivatives are based on their anti-microbial, anti-viral, anti-plaque, antiparasitic, anti-proliferative, and anti-inflammatory effects [7]. KA is used as a skin whitening agent in many countries [8]. Indeed, in a previous study, Mannich base, a derivative of KA, was found to actively inhibit melanin production in melanoma cells, suggesting potential applications as a therapeutic agent for melanoma [9]. KA is also widely used as a food additive owing to its anti-bacterial and

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anti-viral activities [10]. Previous studies have shown that KA has significant anti-microbial properties; its anti-bacterial activity against gram-negative bacteria is higher than that against grampositive bacteria [11]. The bacteriostatic mechanism of KA is mainly caused by obvious damage to cell membrane integrity. KA causes changes in the zeta potential of bacterial cells, thereby disrupting the subcellular localization of some proteins, resulting in bacterial inactivation [12]. Previous studies have shown that KAgrafted-chitosan oligosaccharides can enhance the inhibitory effects of chitosan oligosaccharides on the growth of Staphylococcus aureus, Escherichia coli, Streptococcus pyogenes, and Salmonella typhimurium [13]. To date, the anti-bacterial effects of KA in the preservation of boar semen and the mechanism of bacterial inactivation by KA have not been determined. Therefore, in this study, we investigated the effects of KA on sperm quality parameters, bacterial concentrations, bacterial compositions, Western blot, total anti-oxidant capacity (T-AOC), reactive oxygen species (ROS) content, and malondialdehyde (MDA) content during liquid preservation of boar semen at 17
C. We also evaluated the effects of KA on IVF parameters, spermeoocyte binding and embryonic development in vitro. 2. Materials and methods 2.1. Chemicals Unless otherwise stated, all chemicals used in this study were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Animals Procedures involving animal experiments were approved by the Animal ethics committee (Northwest A&F University, Yangling, Shaanxi). A total of 12 healthy and sexually mature Duroc boars aged 2e4 years and selected according to normal semen quality and proven fertility were used in this study. Boars were fed at Domestic Animal Improving Station (Shaanxi Province, China). All boars were housed in buildings with stable conditions of automatic controlled temperature and humidity and were fed an adjusted commercial diet. 2.3. Semen samples and processing The semen samples were collected using the gloved-hand method once a week and filtered through three layers of gauze to remove gel particles. Pre- and post-sperm-rich fractions were discarded, and only sperm-rich fraction of the ejaculate was collected for analysis. Only semen samples that met the following quality requirements were used:  5.0  108/mL sperm concentration,  80% motile sperm, and 80% normal morphology [14]. The semen samples were diluted with Beltsville thawing solution (BTS) (37.15g glucose, 6.00g Sodium citrate, 1.25 g/L EDTA, 1.25 g/L Sodium bicarbonate, 0.75 g/L Potassium chloride) containing different concentrations of KA, and set to an optimum concentration (5.0  107 sperm cells/mL) using optical density with a calibrated spectrophotometer (Shanghai Spectrophotometer Co., Ltd., Shanghai, China). Immediately after dilution, the diluted semen samples were slowly cooled to 17
C in 12 h, and then the cooled semen samples were transferred to the incubator within 24 h at 17
C. 2.4. Experimental designs In this study, we carried out of two experiments (Fig. 1).

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2.4.1. Experiment I: effects of different concentrations of KA on sperm quality parameters and bacterial concentrations Boar semen samples were diluted with BTS, containing different concentrations of KA (0, 0.02, 0.04, 0.06, 0.08 and 0.10 g/L) at 17
C during liquid storage, and then assessed the sperm motility, mitochondrial membrane potential, acrosome integrity, plasma membrane integrity, and bacterial concentrations within 1e5 days. 2.4.2. Experiment II: effects of 0.04 g/L KA and 0.25 g/L gentamycin supplementation to boar semen dilution on sperm quality parameters, sperm capacitation, bacterial concentrations, bacterial compositions, Western blot, T-AOC ability, ROS content, MDA content, IVF parameters, spermeoocyte binding, and embryonic development In experiment 2, we measured sperm quality parameters, sperm capacitation, bacterial concentrations, bacterial compositions, Western blot, T-AOC ability, ROS content, MDA content, IVF parameters, spermeoocyte binding, and embryonic development. 2.5. Assessment of sperm motility, mitochondrial membrane potential, acrosomal integrity, and plasma membrane integrity The percentage of motile sperm was measured using a computer-assisted semen analysis (CASA) system (Hamilton Thorne Research, Beverly, MA, USA), as reported by Bucci et al. [15], Briefly, a 10-mL aliquot of semen sample was placed on a slide, covered with a coverslip before pre-warming for 30 min at 37
C in a water bath. Five separate fields for each semen sample were randomly selected to assess sperm motility at 400  magnification. Mitochondrial membrane potential was assessed using a specific kit JC-1 (5,50 ,6,60 -tetrachloro-1,10,3,30 -tetraethylbenzimidazolyl carbocyanine iodide) (Solarbio Science & Technology Co, Beijing, China) as described in a previous study [16]. Briefly, 500 mL of semen samples (1  106 spz/mL) were incubated with 5 mL of JC-1 (1 mg/mL) at 37
C for 30 min in dark and 10,000 sperms per biological replicate (five replicates per group) were analyzed by flow cytometry. The percentage of spermatozoa with reacted or damaged acrosomes was assessed after staining the sperm with peanut agglutinin conjugated with fluorescein isothiocyanate (FITC-PNA) and 40 ,6-diamidino-2-phenylindole (DAPI) double staining methods as reported by Fazeli et al. [17]. Briefly, sperm samples were smeared onto glass slides, naturally air-dried, and fixed with methanol for 15 min at 37
C. Subsequently, semen samples were washed three times with phosphate-buffered saline (PBS), and then incubated with FITC-PNA (10 mg/mL) and DAPI (6 mg/mL) for 30 min at 37
C in dark. The sperm plasma membrane integrity was assessed by SYBR-14 and propidium iodide (PI) (LIVE/ DEAD Sperm Viability Kit; Invitrogen, Japan) staining according to the manufacturer's instructions. Briefly, sperm samples were incubated with SYBR14 (final concentration: 100 nM) at 37
C for 10 min and then with propidium iodide (PI) (final concentration: 15 mM) for 5min.The results are expressed as the final percentage of motile sperm (Fig. 2A), mitochondrial membrane potential (Fig. 2B), acrosomal integrity (Fig. 2C), and plasma membrane integrity (Fig. 2D). 2.6. Evaluation of bacterial growth and bacterial compositions To evaluate the anti-bacterial effects of KA in diluted boar semen, the bacterial concentrations and compositions were determined. On each experiment day, the bacterial concentration in each group was evaluated using LB agar plates as described by Bussalleu et al. [18]. Briefly, the semen samples were diluted several fold with Ringer's solution, and then the diluted samples were seeded onto plates. Subsequently, the plates were incubated at

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Fig. 1. Flow chart of the experimental design and procedure. KA, kojic acid.

37
C for 72 h in an incubator under aerobic conditions. The plates with 300 or more colonies or with 15 or fewer colonies were discarded. The plates with 300 or more colonies or with 15 or fewer colonies were discarded. The results are expressed as mean ± standard deviation (SD). After 5 days of preservation, 16S rDNA analysis genes were used to determine bacterial species in each group as reported by Liu et al. [19]. The results of specific bacterial species at the phylum, order, and genus levels are expressed as mean ± SD.

Following treatment, the sperm samples for western blotting were lysed in lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mg/mL pepstatin, 1 mg/mL aprotinin, 1 mg/mL leupeptin, and 1 mM PMSF). Western blotting was performed as our previously described methods [20]. The following antibodies were used in the Western blot assay: Superoxide dismutase 1 (SOD1) (Proteintech Group, Chicago, USA), Superoxide dismutase 2 (SOD1) (Proteintech Group, Chicago, USA), Glutathione peroxidase 5 (Gpx5) (Proteintech Group, Chicago, USA), Catalase (CAT) (Proteintech Group, Chicago, USA), Bax (Proteintech Group, Chicago, USA), Caspase-3 (Proteintech Group, Chicago, USA), and Bcl-2 (Proteintech Group, Chicago, USA).

and MDA content were evaluated using the T-AOC Assay Kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China), Active Oxygen Detection Kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China), and MDA Test Kit (Solarbio Science & Technology Co, Beijing, China), respectively. Briefly, the T-AOC ability and MDA content were evaluated using a spectrophotometer (Shanghai Spectrophotometer Co., Ltd., Shanghai, China) according to the instructions provided with the kit. Briefly, the extracts of sperm were prepared by sonication (20 KHz, 750 W, operating at 40%, on 3 s, off 5 s, 5 cycles) in ice-cold buffer (50 mM Tris-HCl (pH 7.5), 5 mM EDTA, and 1 mM DTT). Supernatants were collected and mixed with the T-AOC reaction buffer. The T-AOC ability of semen sample is expressed as U/mL. To evaluate the MDA content, the lysed cells were centrifuged (12,000g, 10 min) to remove debris, and the supernatant was collected and mixed with the MDA reaction buffer. The MDA content of semen sample is expressed as nmoL/mL. The ROS content in sperm sample was evaluated using DCFH2-DA, according to the instructions provided with the kit. Briefly, 1000 mL of semen sample was centrifuged at 800g for 5 min and washed twice with PBS. Subsequently, the semen samples were incubated with 20 mM DCFH2-DA working solution at 37
C in dark for 20 min. The fluorescence intensity (indicating ROS content) was measured using a multifunctional fluorescence microplate reader at Ex/ Em ¼ 485/525 nm.

2.8. Evaluation of T-AOC ability, ROS content, and MDA content

2.9. Oocyte collection and in vitro maturation (IVM)

2.7. Western blotting

On experimental days 1, 3, and 5, the T-AOC ability, ROS content,

The oocytes used in this study were prepared according to a

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Fig. 2. Sperm motility was detected by CASA (A). Standard of flow cytometry analyses for evaluating sperm mitochondrial membrane potential (B). Acrosome integrity was detected using FITC-PNA and DAPI double staining (C). Plasma membrane integrity was detected using SYBR14 and propidium idodide (PI) double staining (D). Scale bar: 10 mM.

method as described by previously study [21]. Ovaries were obtained from maturing gilts (Duroc  Large White  Landrace) at a local slaughterhouse and transported to the laboratory within 2 h after collection in physiological saline at 37
C. Collection of cumulus-oocyte complexes (COCs) was carried out using an 18gauge needle. Subsequently, the COCs were washed three times, and then intact COCs were selected for maturation in IVM medium. After 42e44 h of IVM culture, the COCs were denuded by gently pipetting with 0.1% hyaluronidase and mature oocytes were selected for the subsequent experiments. 2.10. Assessment of sperm capacitation and IVF parameters Sperm capacitation following the method as reported in a previous study [22]. After 5 days of liquid preservation, semen samples were centrifuged (1500g, 10 min) and transferred to capacitation medium (CM) for 30 min in a humidified incubator at 38.5
C with an atmosphere containing 5% CO2 in air. Immediately after sperm capacitation, changes in sperm capacitation were evaluated by staining with chlortetracycline (CTC). The results are expressed according to three acrosome staining patterns as our previously described methods [23]. After sperm capacitation and culture of oocyte for maturation, 50 COCs were transferred to 500 mL of CM containing different concentrations of sperm, and then incubated in an atmosphere of 5% CO2 at 38.5
C. After 6 h of co-incubation of sperm and oocytes, oocytes were washed three times with D-PBS buffer and

transferred to PZM-3 medium [24] under mineral oil in an atmosphere containing 5% CO2 at 38.5
C for 12 h. Next, oocytes were fixed in 12.5% paraformaldehyde for 30 min, stained with Hoechst 33,342 (10 mg/mL) for 10 min and examined using a phase contrast microscope (Nikon, Tokyo, Japan) at 400  magnification to evaluate IVF parameters, including penetration rates, monospermy rates, and total efficiency. 2.11. Assessment of spermeoocyte binding For the spermeoocyte binding assays, spermeoocyte binding was performed as described by Miao et al. [25], with some modifications. Spermeoocyte co-incubation was carried out as described above for microdrop IVF preparations. After incubation for 6 h, oocytes were washed three times with PBS containing 0.1% PVA, stained with Hoechst 33,342 (10 mg/mL) for 10 min, and examined by phase-contrast microscopy (Nikon, Tokyo, Japan) at 400  magnification for the spermeoocyte binding assays. 2.12. Assessment of embryonic development For embryonic development assays, embryo culture was performed as described by Uh et al. [22]. Spermeoocyte co-incubation was carried out as described above for microdrop IVF preparations. After incubation for 6 h, oocytes were washed with D-PBS, transferred to PZM-3 medium, and incubated at 38.5
C for 168 h in an atmosphere containing 5% CO2 in humidified air. The oocyte

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cleavage and blastocyst formation rates were assessed by microscopy (Nikon, Tokyo, Japan) after 48h and 168 h of culture. Oocytes were classified according to the cleavage and blastocyst rates, and the results are presented as the percentage of total number of oocytes per group. 2.13. Statistical analysis All results are presented as mean ± standard deviation (SD). Statistical Package for the Social Sciences software, version 21 (SPSS Inc., Chicago, IL, USA) was used for data analyses. All the variables were first tested for normality (ShapiroeWilk test) and homoscedasticity (Levene test). When necessary, data (x) were transformed by calculating the arcsine square root (arcsin √x). The effects of treatments and storage at 17
C on sperm quality parameters, bacterial concentrations, bacterial compositions, T-AOC ability, ROS content, MDA content, sperm capacitation, IVF parameters, and embryonic development were tested using a linear mixed model, followed by Sidak's post-hoc test for pair-wise comparisons. The treatment was the fixed-effects factor, each ejaculate was the random-effects factor, and storage period was the intra-subject factor. Each test parameter after culture, including sperm quality parameters, bacterial concentrations, bacterial compositions, TAOC ability, ROS content, MDA content, sperm capacitation, IVF parameters, and spermeoocyte binding, was the variable. The embryonic development, including oocyte cleavage and blastocyst cleavage, were analyzed using a Chi-square test. Experiments I and II were evaluated separately and the results with P values of less than 0.05 were considered significant. 3. Results 3.1. Experiment I 3.1.1. Effects of different concentrations of KA on sperm quality parameters Changes in sperm quality parameters in all the groups are shown in Fig. 3. After 5 days of liquid storage, all sperm quality parameters were lower than those at the beginning of the experiment (P < 0.05). Notably, sperm motility and plasma membrane integrity were higher in the 0.04 g/L KA group than in the other groups (P < 0.05). Acrosome integrity was similar for all treatment groups, but higher in the treatment groups than in the control group (P < 0.05). In addition, mitochondrial membrane potential integrity was significantly higher in the 0.02 g/L and 0.04 g/L KA groups than in the control groups (P < 0.05). Overall, these results suggested that 0.04 g/L is the optimum concentration of KA for enhancement of sperm quality parameters. 3.1.2. Effects of different concentrations of KA on bacterial concentrations The effects of KA concentration on bacterial concentrations are shown in Fig. 4A. In all the groups, an increase in bacterial concentration was observed from the beginning of the experiment to the end of the storage period (P < 0.05). As the KA concentration increased, the bacterial concentration was gradually decreased during liquid storage, and significant differences were observed for all groups (Control: 7.89 ± 0.49, 0.02 g/L KA: 5.93 ± 0.37, 0.04 g/L KA: 4.18 ± 0.56, 0.06 g/L KA: 3.66 ± 0.71, 0.08 g/L KA: 3.51 ± 0.39, 0.10 g/L KA: 3.21 ± 0.49) (except 0.06 g/L KA and 0.08 g/L KA groups) on experimental day 5 (P < 0.05). These findings indicated that KA at high concentrations exhibited strong anti-bacterial effects during semen liquid storage at 17
C.

3.2. Experiment II 3.2.1. Effects of KA and gentamycin on bacterial concentrations and bacterial compositions The effects of KA and gentamycin on bacterial concentrations are shown in Fig. 4B. In all the groups, an increase in bacterial concentration was observed from the beginning of the experiment to the end of the storage period (P < 0.05). After 5 days of preservation, the bacterial concentrations of the KA group (4.21 ± 0.61) were significantly higher than those of the gentamycin group (3.68 ± 0.46) (P < 0.05). This indicated that the anti-bacterial effect of 0.04 g/L KA was worse than that of 0.25 g/L gentamycin. The broad-spectrum anti-bacterial effects of KA are still unknown; thus, we further evaluated the occurrence of bacterial species in diluted semen in the presence or absence of KA by sequencing the bacterial 16S rDNA during storage. The overall microbial compositions in the three groups differed at the phylum, order, and genus levels (Fig. 4C-E). At the phylum level, 10 phyla were detected in diluted semen. In particular, there were four phyla with a quantity of 0.5% in each group, i.e., Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Proteobacteria and Actinobacteria of the control group were significantly higher than those of the 0.04 g/L KA and gentamycin groups (P < 0.05), and Firmicutes and Bacteroidetes of the 0.04 g/L KA and gentamycin groups were significantly higher than those of the control group (P < 0.05). At the order level, the changes of bacterial compositions in treatment groups were similar to those at the phylum level. At the genus level, 29 genera were identified. The percentage of 10 of these genera was 0.5% (Fig. 4E). In the 0.04 g/L KA group, six genera, i.e., Enterobacter, Streptococcus, Shewanella, Staphylococcus, EscherichiaShigella, and Bifidobacterium, were significantly decreased compared with those in the control group (P < 0.05). In addition, Bacillus and Bacteroides of the 0.04 g/L KA group were significantly increased (P < 0.05). However, in the gentamycin group, nine genera, i.e., Enterobacter, Streptococcus, Shewanella, EscherichiaShigella, Bifidobacterium, Bacillus, Bacteroides, Atopostipes, and Staphylococcus, were significantly decreased compared with those in the control group (P < 0.05). These results indicated that KA does not have broad-spectrum anti-bacterial activity, but instead only inhibited the growth of specific bacterial species. 3.2.2. Effects of KA and gentamycin on sperm quality parameters and sperm capacitation The results of KA and gentamycin on sperm quality parameters and sperm capacitation are shown in Fig. 5. After 5 days of liquid storage, the sperm quality parameters of the KA and gentamycin groups were significantly higher than those of the control group (P < 0.05), and the sperm quality parameters (except plasma membrane integrity) were not significantly difference between the KA and gentamycin groups. The changes of sperm capacitation are shown in Fig. 5E. The percentage of spermatozoa with CTC staining fluorescent pattern B (capacitated sperm) in the KA and gentamycin groups was significantly increased compared with that in the control group (P < 0.05), and the pattern B (capacitated sperm) in the gentamycin group was significantly higher than that in the KA group (P < 0.05). These results indicated that KA and gentamycin could promote sperm capacitation. 3.2.3. Effects of KA and gentamycin on anti-oxidant- and apoptosisrelated proteins in sperm On day 5 of preservation, the levels of anti-oxidant- and apoptosis-related proteins in sperm are shown in Fig. 6. In comparison with that in the control group, the expression of SOD1, SOD2, and CAT in sperm in the KA and gentamycin groups was significantly enhanced (P < 0.05). However, the expression of anti-

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Fig. 3. Changes in sperm quality parameters during liquid preservation in semen dilution containing different concentrations of KA. A. Sperm motility. B. Mitochondrial membrane potential. C. Acrosome integrity. D. Plasma membrane integrity. Results are represented as mean ± SD (n ¼ 5 per group). Different superscripts (aec) mean significant differences (P < 0.05). KA, kojic acid.

oxidant-related protein GPx5 was not significant between the

control and treatment groups, and the expression of anti-oxidant-

Fig. 4. Differences in bacterial concentrations and bacterial compositions in semen dilution containing gentamycin and different concentrations of KA. A. Bacterial concentration. B. Bacterial concentration. C. Bacterial compositions at the phylum level. D. Bacterial compositions at the order level. E. Bacterial compositions at the genus level. Results are represented as mean ± SD (n ¼ 5 per group). Different superscripts (aee) mean significant differences (P < 0.05). *P < 0.05; **P < 0.01; ***P < 0.001. Cont: control group; KA: 0.04 g/L KA group; Gentamycin, 0.25 g/L gentamycin group. KA, kojic acid.

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Fig. 5. Differences in sperm quality parameters and sperm capacitation among control group and treatment groups. A. Sperm motility. B. Mitochondrial membrane potential. C. Acrosome integrity. D. Plasma membrane integrity. E. sperm capacitation; Pattern A: Uncapacitated sperm; Pattern B: Capacitated sperm; Pattern AR: Acrosome-reacted sperm. Results are represented as mean ± SD (n ¼ 5 per group). Different superscripts (aec) mean significant differences (P < 0.05). *P < 0.05; **P < 0.01; ***P < 0.001. Cont: control group; KA: 0.04 g/L KA group; Gentamycin, 0.25 g/L gentamycin group. KA, kojic acid.

related proteins in sperm in the KA and gentamycin group was not significant. In comparison with that in the control group, the expression of Caspase-3 and Bax in sperm in the KA and gentamycin groups was significantly inhibited (P < 0.05), and the antiapoptosis-related protein Bcl-2 in sperm in the KA and gentamycin groups was significantly enhanced (P < 0.05). These results confirmed that KA could inhibit apoptosis of sperm and enhance anti-oxidant capacity of sperm in vitro. 3.2.4. Effects of KA and gentamycin on T-AOC ability, ROS content, and MDA content After 5 days of preservation, the results of T-AOC ability, ROS

content, and MDA content are shown in Fig. 7. In comparison with that in the control group, the T-AOC ability was significantly enhanced in the 0.04 g/L KA and gentamycin groups (P < 0.05). Furthermore, the results showed that the addition of KA and gentamycin significantly reduced the ROS and MDA content compared with that in the control group (P < 0.05). 3.2.5. Effects of KA and gentamycin on IVF parameters Polyspermic fertilization is a major factor that prevents embryonic development in swine; thus, we measured IVF parameters to identify a suitable spermeoocyte culture concentration, and the results demonstrated that the suitable spermeoocyte culture

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Fig. 6. Western blot analysis of antioxidant- and apoptosis-related proteins in sperm on the 5th day storage. A. Antioxidant-related proteins expression level in sperm. B. Apoptosisrelated proteins expression level in sperm. Results are represented as mean ± SD (n ¼ 5 per group). *P < 0.05; **P < 0.01. Cont: control group; KA: 0.04 g/L KA group; Gentamycin, 0.25 g/L gentamycin group. KA, kojic acid.

concentration was 2.0  104 spz/mL in the KA group (Fig. S1). The results of IVF parameters were shown in Fig. 8. Among the three groups, the significantly higher penetration rate was observed in the gentamycin group than that in the other groups (P < 0.05). However, significantly higher proportion of monospermy and total efficiency were observed in the KA group than those in the other groups (P < 0.05). These results suggested that KA could enhance monospermy rate and total efficiency (Figs. 5 and 8), and the gentamycin group containing a high ratio of capacitated sperm resulted in polyspermic fertilization compared with that in the KA group (P < 0.05). 3.2.6. Effects of KA and gentamycin on spermeoocyte binding and embryonic development The differences of spermeoocyte binding, cleavage rate, and blastocyst rate are shown in Fig. 9. Among the three groups, the significantly higher number of sperms attached to oocytes were observed in the gentamycin group p (32.01 ± 4.97) than that in the other groups (Control: 15.88 ± 3.26, 0.04 g/L KA: 26.39 ± 4.99) (P < 0.05). However, significantly lower cleavage (25.59 ± 3.09) and blastocyst rates (10.761 ± 2.61) were observed in the gentamycin group than those in the KA group (cleavage rates: 32.27 ± 4.63; blastocyst rates: 15.69 ± 4.09) (P < 0.05). When comparing the two groups, including control group and gentamycin group, KA resulted in the best IVF results, cleavage rates, and blastocyst rates were significantly higher in the KA group (P < 0.05). Thus, these findings indicated that KA can enhance the number of sperms attached to oocytes and increase the cleavage and blastocyst rates. 4. Discussion Antibiotics have been extensively used to inhibit bacterial growth in semen liquid preservation. Nevertheless, because of concerns regarding the negative effects of antibiotics in the pig industry, it is essential to identify effective substitutes for antibiotics. This study was firstly conducted to evaluate the effect of KA on sperm quality and anti-bacterial activity during liquid storage at 17
C. The results of this study showed that 0.04 g/L KA can enhanced sperm quality parameters, sperm capacitation, anti-

oxidant-related protein expression, spermeoocyte binding, and embryonic development, inhibited bacterial growth and apoptosisrelated protein expression, and altered bacterial compositions. High bacterial loading has deleterious effects on sperm quality parameters, including sperm motility, acrosome integrity, plasma membrane integrity, and mitochondrial membrane potential integrity [26,27]. The direct interaction between bacteria and sperm quality depends on specific toxins released by bacteria, which results in affecting plasma membrane integrity, altering mitochondrial membrane potential, and impairing motility [28e31]. The experiment I we performed, together with bacterial compositions of experiment II, was aimed to evaluate the antibacterial effect of KA on sperm quality and bacterial growth. The results showed that 0.04 g/L KA enhanced sperm quality and inhibited bacterial growth, although only some bacterial genera, such as Enterobacter, Streptococcus, Shewanella, Escherichia-Shigella, Staphylococcus, and Bifidobacterium, were inhibited. In addition, sperm motility and plasma membrane integrity were better in the 0.04 g/L KA group than in the groups treated with higher concentrations of KA. Thus, these results suggested that KA was deleterious to boar spermatozoa in semen dilutions when the KA concentration exceeded 0.04 g/L; accordingly, we selected 0.04 g/L as the optimal concentration of KA. Conversely, acrosome integrity did not differ among all treatment groups. From these findings, we speculated that KA maintained bacterial concentrations within the threshold in the semen dilutions and did not alter sperm acrosome integrity [32,33]. However, further studies are needed to verify this hypothesis. To compare the anti-bacterial effects of KA and gentamicin, in experiment II, we evaluated bacterial concentrations, sperm quality parameters, and sperm capacitation. The results showed that sperm motility, plasma membrane integrity, and mitochondrial membrane potential did not differ between the KA and gentamycin groups. In addition, the bacterial concentrations were higher in the KA group and capacitated sperms were higher in the gentamycin group. These findings suggested that KA maintained bacterial concentrations within the threshold in the semen dilutions and did not alter sperm quality parameters (except plasma membrane integrity), but higher bacterial loading damaged sperm capacitation

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Fig. 7. Differences in T-AOC ability, ROS content and MDA content among control group and treatment groups. A. T-AOC ability. B. ROS content. C. MDA content. Results are represented as mean ± SD (n ¼ 5 per group). *P < 0.05. Cont: control group; KA: 0.04 g/L KA group; Gentamycin, 0.25 g/L gentamycin group. KA, kojic acid.

and promoted sperm apoptosis. One of the most interesting results of experiment II is changes in plasma membrane integrity between the KA and gentamycin groups. In this study, compared with that of the gentamycin group, the sperm plasma membrane integrity was significantly increased in the KA group. As evidenced in previous studies, several factors affect sperm plasma membrane integrity, including oxidative stress, bacterial concentrations, bacterial byproducts, osmotic pressure, and sperm metabolism products [34e36]. Therefore, two main mechanisms could account for this phenomenon. One possible reason for this discrepancy is that the anti-bacterial effect of KA is worse than that of the gentamycin group, resulting in the production of toxins released by bacteria is significantly higher in KA group than in gentamycin group; thereby, decreasing sperm plasma membrane integrity in the KA group. Another explanation could involve the slight toxic effect of KA; KA might have interacted with phospholipid bilayers and undermined

membrane structure and integrity during liquid storage, but the sperm plasma membrane integrity was significantly higher in the 0.04 g/L KA group than in the other KA groups, suggesting that KA only exerted minor toxicity on sperm and that it could be applied to semen dilution for liquid storage. Bacterial loading and oxidative stress are the key factors affecting perm quality. In this study, we observed an apparent relationship between bacterial growth and sperm ROS content during semen preservation. Bacterial byproducts, including a-hemolysin, lipopolysaccharide, and Shiga-like products, result in sperm oxidative stress and oxidative mitochondrial injury [36,37]. As reported in a previous study, ROS, including hydrogen peroxide, superoxide anion, hydroxyl radical, hypochlorite radical, and singlet oxygen, are formed as normal byproducts of physiological processes in sperm when incubated under aerobic conditions [38]. High levels of ROS or excessively low levels of ROS have deleterious

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Fig. 8. Differences in penetration rate, monospermy rate and total efficiency of control group and treatment groups. A. Immunofluorescent analysis of penetration rate, monospermy rate and total efficiency; a: Unfertilized oocyte; b, c: Polyspermic oocyte. d, e: Two-pronuclear. B. Penetration rate (number of oocytes penetrated/number of inseminated oocytes). C. Monospermy rate (number of oocytes penetrated by one spermatozoon/number of penetrated oocytes). D. Total efficiency (number of oocytes penetrated by one spermatozoon/number of inseminated oocytes). 248e253 oocytes in each group. Results are represented as mean ± SD (n ¼ 5 per group). *P < 0.05. Cont: control group; KA: 0.04 g/L KA group; Gentamycin, 0.25 g/L gentamycin group. PN, pronucleus; PB, polar body; KA, kojic acid. Scale bar: 100 mM.

effects on sperm quality. When ROS are present at low concentrations, cellular functions are disrupted because ROS regulate many

biochemical reactions. However, when ROS are present at high concentrations, severe injury can occur to various cellular

Fig. 9. Differences in sperm-oocyte binding and embryonic development among control group and treatment groups. A. Sperm-oocyte binding; Scale bar: 50 mM. 246e252 oocytes in each group. B. Cleavage rate; Scale bar: 400 mM. 497e513 oocytes in each group. C. blastocyst rate; Scale bar: 50 mM. 497e513 oocytes in each group. Results are represented as mean ± SD (n ¼ 5 per group). *P < 0.05; **P < 0.01. Cont: control group; KA: 0.04 g/L KA group; Gentamycin, 0.25 g/L gentamycin group; KA, kojic acid.

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components; indeed, excessive ROS can damage DNA integrity, reduce plasma membrane integrity, disrupt mitochondrial membrane potential, and activate pro-apoptotic caspases (caspase-9 and caspase-3), leading to apoptosis. In contrast, the optimum concentration of ROS has positive effects on sperm functions, including sperm capacitation, acrosome reaction, cAMP production, hyperactivation, and mitochondrial oxidative phosphorylation, which are required for fertilization [39]. In previous studies, KA has been used an important agent to scavenge ROS, including hydrogen peroxide, superoxide anion, hydroxyl radical, and singlet oxygen [40,41]. In our study, the results showed that 0.04 g/L KA significantly enhanced the T-AOC ability and reduced ROS and MDA content compared with those in the control group. Compared with those in the gentamycin group, the bacterial concentrations were significantly elevated in the KA group, but the T-AOC ability and ROS and MDA content were not different. The imbalance between the production of ROS and the activity of anti-oxidant defense systems is a main cause of oxidative stress [42]. In this context, we concluded that KA enhanced sperm quality parameters, mainly through antioxidant effect of KA itself to maintain the T-AOC ability, and then inhibited the overproduction of ROS and MDA. In swine, there is a high incidence (>50%) of polyspermy, representing a major obstacle in embryonic development when oocytes are fertilized with excessive capacitated spermatozoa [43]. The reduced concentrations of spermatozoa used for IVF indeed decrease polyspermy, but also reduce penetration rates [44]. Therefore, application of an optimal concentration of sperm fertilized with oocytes is extremely important to reduce polyspermy and obtain reliable results of embryonic development in vitro. Our results showed that optimal spermeoocyte culture concentration was 2.0  104 spz/mL in the KA group. However, with respect to the IVF parameters, the results showed that the monospermy rate and total efficiency were significantly higher in the KA group than in the other groups. These findings indicated that higher capacitated sperm of gentamycin group decreased the monospermy rate and total efficiency, and subsequently blocked embryonic development. As stated above, sperm quality is affected by bacteria, which can lead to fertilization failure. Thus, we further assessed spermeoocyte binding and embryonic development. Compared with those of the control group, KA treatment significantly increased the number of sperms attached to oocytes, and the rate of cleavage and blastocyst, indicating that KA promoted spermeoocyte binding, and cleavage and blastocyst rates. In the control group, reduced number of sperms attached to oocytes, suggesting that bacteria and bacterial toxins may have reduced the number of high-quality sperm cells for attachment to oocytes. This can be attributed to two main mechanisms accounting. First, the abovementioned toxins released by bacteria may have disrupted sperm function, resulting in failure of sperm capacitation. In a previous study, membrane proteins, such as spermeoocyte binding proteins on the sperm head, ZP-binding molecules, and multimeric ZP recognition complexes, were found to be associated with a highly redundant process for complete penetration [45e47]. Thus, we speculated that spermeoocyte recognition proteins may also be damaged or masked by bacteria and bacterial byproducts, thereby reducing spermeoocyte recognition and penetration rates, and subsequently disrupting embryonic development. 5. Conclusion In conclusion, our findings demonstrated that KA enhanced sperm quality, sperm capacitation, anti-oxidant-related protein expression, T-AOC ability, IVF parameters, sperm attachment to oocytes, and embryonic development, as well as inhibited bacterial growth, apoptosis-related protein expression, ROS content, and

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