Infertility is a prevalent concern that affects humans and animals worldwide. The World Health Organization estimated that 15% of couples worldwide have fertility problems, and male infertility accounts for approximately 50% of all infertility cases in the animal industry (Peddinti et al., 2008; Park et al., 2013). Moreover, infertility can cause huge economic losses and the collapse of the animal industry (Watson, 2000). As it is important to investigate male fertility, in vitro studies on male fertility have been conducted in various fields (de Angelis et al., 2017; González-Rodríguez et al., 2018). Furthermore, studies related to fertility have been conducted on various animals (Jamsai and O’Bryan, 2011; Fortes et al., 2013). Especially in pigs, studies have been conducted on reproduction (Zigo et al., 2020). In this study, we used boar spermatozoa to investigate reproduction and male fertility. Because they have greater physiological similarities with humans than other domestic animals, pigs can be used as organ donors for xenotransplantation or the production of transgenic animals (Romar et al., 2016; Romar et al., 2019). Thus, pigs are among the best animal models for studying male fertility.

Freshly ejaculated spermatozoa encounter different environments when passing through the female reproductive tract. Physiological changes, known as capacitation, occur as a result of various factors in the female reproductive tract (de Lamirande et al., 1997). In addition, sperm motility is a major factor that determines sperm quality and plays a key role in fertilization. However, sperm motility deteriorates over a long period (Alavi and Cosson, 2005; You et al., 2017). Capacitation causes spermatozoa to change their motility and physiology. During capacitation, sperm motility, kinematic motion change, and the membrane of the sperm head are also destabilized. Consequently, capacitated spermatozoa can exclusively undergo the acrosome reaction, which allows them to penetrate the zona pellucida (Austin, 1952; Baker et al., 2012; Kwon et al., 2014; Bae et al., 2019).

Capacitation is an in vivo phenomenon (Austin, 1951; Chang, 1951); however, the current knowledge of capacitation has been obtained from in vitro studies. Various trials have been performed to establish in vitro capacitation methods for reproductive studies, and developing a more accurate and standard protocol may be important for successfully inducing capacitation (Kidder, 2014; Rubessa et al., 2019). Although many studies have been conducted to develop an optimal protocol to induce the capacitation of boar spermatozoa, most studies have focused on identifying the appropriate chemical compounds needed to induce the capacitation of boar spermatozoa in vitro (Byrd, 1981; Banerjee and Chowdhury, 1995). Therefore, a new approach is required to ensure optimal capacitation. Few studies have investigated the correlation between incubation time and capacitation, and in general, the incubation time investigated is under 60 min (Herreros et al., 2005; Dapino et al., 2006). Therefore, the purpose of this study is to evaluate whether there is an effect of incubation time (60, 90, and 120 min) on capacitation induction and sperm functions to elucidate the optimal protocol on incubation time for boar spermatozoa in vitro.

Media and chemicals

The medium was prepared as previously described (Kwon et al., 2015; Bae et al., 2019; Bae et al., 2022). Modified tissue culture medium (mTCM) 199 was used as the basic medium (BM) [3.05 mM D-glucose, 2.92 mM calcium lactate, 0.91 mM sodium pyruvate, 10% fetal bovine serum, and 2.2 g/L sodium bicarbonate (Sigma-Aldrich, St Louis, MO, USA)]. We added 10 μg/mL of heparin to induce sperm capacitation.

Sample preparation

All animal procedures were performed in accordance with the guidelines for the ethical treatment of animals and approved by the Institutional Animal Care and Use Committee of Kyungpook National University. Duroc boars were housed in a facility with controlled temperature (20 ± 5℃) and ventilation in Gyeongsan Swine (Gyeongsan, Korea). Twenty-seven Duroc sperm samples were collected from healthy mature Duroc boars using the gloved-hand technique and then diluted (3 × 10⁹ sperm cells/mL) with a broad extender (Beltsville thawing solution). Each experiment was repeated at least 5 times using randomly mixed 3 semen samples for each replicate. The extended semen was stored at 17℃ and processed within 2 h of collection. Three semen samples were mixed to exclude individual variations. Consequently, the samples were incubated with BM (containing 10 μg/mL heparin) for various times (60, 90, and 120 min) at 37℃ and 5% CO₂ to induce capacitation (Bae et al., 2019; Lee et al., 2022).

Sperm motility and motion kinematics

To evaluate sperm motility and motion kinematics, a computer-assisted sperm analysis (CASA) program (FSA2016, Medical supply, Seoul, Korea) with a CMOS image sensor, a 2048 × 1536 (300 M pixels), 60 frame camera (Medical supply, Seoul, Korea), and a BX43 phase-contrast microscope (Olympus, Tokyo, Japan) with a 10× objective phase-contrast mode were used. A sample of 10 μL (30-40 × 10⁶ cells/mL) was smeared on a preheated (37℃) Makler counting chamber (Sefi-Medical Instruments, Haifa, Israel). At least 250 sperm cells were analyzed in five randomly selected fields for each sample. The obtained images were analyzed to determine total sperm motility (MOT, %), rapid sperm motility (RPD, %), medium sperm motility (MED, %), slow sperm motility (SLW, %), progressive sperm motility (PRG, %), curvilinear velocity (VCL, μm/s), straight-line velocity (VSL, μm/s), average path velocity (VAP, μm/s), linearity (LIN, %), straightness (STR, %), beat-cross frequency (BCF, Hz), mean angular displacement (MAD, degree), wobble (WOB, %), dance (DNC, μm²/sec), dance mean (DNM, μm), and amplitude of lateral head displacement (ALH, μm).

Sperm capacitation status

We utilized the H33258/CTC dual staining method (combined Hoechst 33258/chlortetracycline fluorescence assessment) to assess the capacitation status. The sample was centrifuged at 5,000 × g for 5 min. After removing most of the supernatant, 135 μL of DPBS and 15 μL of H33258 solution (10 μg H33258/mL DPBS) were added to the remaining sample and incubated at room temperature (RT) for 5 min. After 5 min, 250 μL of 2% (w/v) polyvinylpyrrolidone was added to the sample. The sample was then washed by centrifugation at 5,000× g for 5 min. The supernatant liquid was removed from the centrifuged samples, and the pellet was resuspended in 500 μL DPBS and 500 μL CTC solution (20 mM Tris, 130 mM NaCl, and 5 mM cysteine, pH 7.4). After refrigerating the sample for 20 min in the dark, 10 mL of the sample was smeared on a glass slide. For each sample, at least 400 spermatozoa were counted on each slide, and a BX43 microscope (Olympus, Tokyo, Japan) with epifluorescence illumination was used as the excitation (BP 340–380/LP 425)/emission (BP 450–490/LP 515) filter (Olympus, Tokyo, Japan). Finally, samples were evaluated and separated into four groups depending on capacitation status: alive and acrosome-reacted sperm (AR pattern, no fluorescence over the head), live and capacitated sperm (B pattern, green fluorescence in the acrosomal region), alive and non-capacitated sperm (F pattern, green fluorescence over the head), and dead sperm (D pattern, blue fluorescence). Consequently, the ratios of the AR, B, and F patterns were calculated based on live spermatozoa.

Statistical analysis

All data were analyzed by one-way ANOVA using SPSS (ver. 25.0, IBM, Armonk, NY, USA). Tukey’s multiple comparison test was used to compare three groups. Each experiment was performed at least five times. Data are presented as the mean ± SEM. Statistical significance was set at p < 0.05.

The fact that spermatozoa must remain in the female reproductive tract for a certain period to acquire fertility was first reported in 1951 by Colin Russell Austin and Min Chueh Chang in rats and rabbits, respectively (Austin, 1951; Chang, 1951). Austin coined the name “capacitation” in 1952, and it is now referred to as in vivo capacitation (Austin, 1952). Capacitation can also be fulfilled in vitro using appropriate chemical compounds, such as BSA, glycoproteins, and ions (Naz and Rajesh, 2004; Bailey, 2010; Jin and Yang, 2017). To penetrate and fertilize an egg, the sperm must undergo capacitation, which causes many physiological changes in the sperm. As a result, sperm motion kinematics are altered, and capacitated sperm undergo an acrosome reaction; only acrosome-reacted sperm can penetrate the egg (Austin, 1952; Baker et al., 2012; Kwon et al., 2014; Bae et al., 2019). It is important to consider inducing capacitation to achieve successful in vitro fertilization.

Capacitation is an important process that occurs after ejaculation in the female reproductive tract. Various studies have been conducted to develop an optimal protocol for inducing capacitation in boar spermatozoa. However, while various chemical compounds have been identified to induce the capacitation of boar spermatozoa in vitro (Bailey, 2010; Jin and Yang, 2017), few studies have described the correlation between incubation time and capacitation in vitro (Herreros et al., 2005; Dapino et al., 2006). However, it is not clear what is the best incubation time to induce capacitation in vitro for boar spermatozoa. Thus, we performed the present study to determine the optimal incubation time for capacitation in vitro.

Generally, various incubation time is applied to induce capacitation for mouse, bull, human, and ram spermatozoa in vitro in studies related to reproduction (Wattimena, 2006; Ryu et al., 2014; Bae et al., 2019; Sáez-Espinosa et al., 2020). Therefore, the present study was designed to find the optimal incubation time for in vitro capacitation of boar among various incubation times (60 min, 90 min, or 120 min) described in previous studies by analysis of sperm motion parameters and morphological changes. Sperm motility and morphological change are a major factor determining the quality of sperm and have a key role in fertilization. Therefore, analysis of sperm motion parameters and capacitation status are basic approaches in various studies related to reproduction based on capacitation (Bae et al., 2020; Bae and Kwon, 2020; Hwang et al., 2021). Additionally, it has been reported that the sperm motility is change based on incubation time (Alavi and Cosson, 2005; You et al., 2017).

In the present study, we used the CASA program to measure a series of sperm motility and motion kinematic parameters. Among the sperm motility and motion kinematics parameters investigated, MOT (%), RPD (%), PRG (%), VCL (μm/s), and VAP (μm/s) were significantly decreased in a time-dependent manner (p < 0.05; Table 1 and Fig. 1). MED (%), SLW (%), VSL (μm/s), LIN (%), STR (%), BCF (Hz), MAD (degree), WOB (%), DNC (μm²/sec), DNM (μm), and ALH (μm) showed no significant changes over time (Table 1). Furthermore, we conducted H33258/CTC dual staining to determine the effects of incubation time on the capacitation status. The results showed that the AR pattern did not change significantly in a time-dependent manner (Fig. 2A). Pattern B showed no significant time-dependent changes (Fig. 2B). In addition, the F-pattern showed no significant changes in a time-dependent manner (Fig. 2C). The results seem to indicate that the incubation time does not affect the capacitation status of boar spermatozoa up to at least 120 min, while the motility and kinematics decrease in an incubation time-dependent manner. Taken together, it is suggested that an increase in incubation time of more than 60 min has a detrimental effect on sperm motility and motion kinematic parameters. Therefore, we suggest that 60 min may be the best incubation time to induce capacitation without negative effects on sperm motility and motion kinematics of boar spermatozoa in vitro. Here, we present our understanding of the effect of incubation time on sperm functions, such as sperm motility, motion kinematic parameters, and capacitation status, in a time-dependent manner and provide better evidence for guidelines to induce capacitation. Therefore, we hope that our results clarify the effects of incubation time and can be used as basic data in experiments related to fertility and reproduction.

Table 1. Sperm motility and kinematics following incubation time.

	Incubation time (min)
	60	90	120
MOT (%)	78.8 ± 1.42a	72.28 ± 2.23a,b	66.91 ± 1.47b
RPD (%)	67.37 ± 1.72a	60.92 ± 2.6a,b	56.68 ± 1.55b
MDM (%)	8.3 ± 0.38	8.07 ± 0.42	6.79 ± 0.33
SLW (%)	3.12 ± 0.26	3.12 ± 0.3	3.38 ± 0.33
PRG (%)	75.68 ± 1.41a	68.99 ± 2.4a,b	63.46 ± 1.28b
VCL (μm/s)	83.16 ± 2.72a	76.28 ± 5.49a,b	67.17 ± 2.06b
VSL (μm/s)	41.3 ± 1.75	37.44 ± 1.65	35.69 ± 1.46
VAP (μm/s)	59.51 ± 2.01a	53.3 ± 3.25a,b	48.52 ± 1.97b
LIN (%)	41.38 ± 1.28	37.62 ± 0.64	37.04 ± 1.23
STR (%)	69.41 ± 1.05	70.7 ± 1.59	73.53 ± 0.65
BCF (Hz)	5.69 ± 0.3	4.99 ± 0.26	4.79 ± 0.14
MAD (degree)	58.85 ± 2.89	52.89 ± 2.4	52.27 ± 2.62
WOB (%)	71.74 ± 1.01	70.42 ± 1.3	72.37 ± 1.02
DNC (μm2/s)	279.75 ± 20.9	243.65 ± 40.56	174.58 ± 10.59
DNM (μm)	8.02 ± 0.44	8.1 ± 0.78	6.9 ± 0.25
ALH (μm)	3.28 ± 0.15	3 ± 0.28	2.53 ± 0.1

Sperm motility and kinematics are presented as mean ± SEM, n = 5. Different superscripts (^a,b) within the same row indicate significant differences (p < 0.05) in the one-way ANOVA. MOT, Total sperm motility (%); RPD, Rapid sperm motility (%); MED, Medium sperm motility (%); SLW, Slow sperm motility (%); PRG, Progressive sperm motility (%); VCL, Curvilinear velocity (μm/s); VSL, Straight-line velocity (μm/s); VAP, Average path velocity (μm/s); LIN, linearity [%, (VSL/VCL) × 100]; STR, Straightness [%, (VSL/VAP) × 100]; BCF, Beat-cross frequency (Hz); MAD, Mean angular displacement (degree); WOB, Wobble [%, (VAP/VCL) × 100]; DNC, Dance [μm²/sec, (VCL × ALH)]; DNM, Mean dance [μm, (ALH/LIN)]; ALH, Mean amplitude of head lateral displacement (μm)..

Figure 1.Sperm motility and kinematics following incubation time. (A) Total Sperm motility. (B) Rapid sperm motility. (C) Progressive sperm motility. (D) Curvilinear velocity (VCL). (E) Average path velocity (VAP). Sperm motility and kinematics are expressed as mean ± SEM, n = 5. Different superscripts (^a,b) within the same row indicate significant differences (p < 0.05) determined by one-way ANOVA.

Figure 2.Sperm capacitation status following incubation time. (A) Patterns of alive and acrosome reacted (AR pattern). (B) Patterns of alive and capacitated (B pattern). (C) Patterns of alive and non-capacitated (F pattern). Values with different superscripts (^a,b) indicate significant differences between the control and treatment groups (p < 0.05). Data were evaluated by one-way ANOVA and represent means ± SEM, n = 9.

In conclusion, boar spermatozoa MOT (%), RPD (%), PRG (%), VCL (μm/s), and VAP (μm/s) significantly decreased in a time-dependent manner. However, capacitation status showed no significant time-dependent differences. Thus, 60 min may be the best incubation time to induce capacitation and maintain sperm motility and motion kinematics. These findings may be used as basic data for inducing capacitation in various studies related to reproduction.

None.

Conceptualization, S-I.J., J-W.B., W-S.K.; methodology, S-I.J., J-W.B., W-S.K.; Investigation, S-I.J., J-H.J., E-J.J., W-J.L., J-M.H., J-W.B., W-S.K. data curation, S-I.J., J-W.B., W-S.K.; writing—original draft preparation, S-I.J., J-W.B., W-S.K.; writing—review and editing, S-I.J., J-W.B., W-S.K.; supervision, W-S.K.; project administration, W-S.K.; funding acquisition, W-S.K.

This study was conducted with the support of the Gyeongsangbuk-do agricultural and fishery R&D activation project.

KNU 2021-207.

Not applicable.

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No potential conflict of interest relevant to this article was reported.