Motile porcine sperms adhere to hydrophilic materials such as glass and plastics. The adsorption of sperms to a hydrophobic poly(dimethylsiloxane) (PDMS) membrane is less compared with that to glass. We investigated the linear velocity (LV) and amplitude of lateral head displacement (ALHD) of motile porcine sperm on glass and PDMS preparations using computer-assisted sperm analysis (CASA). Significant decreases were observed in the 15-min LV (P<0.05) and ALHD (P<0.05) in motile porcine sperm on glass preparations compared with those on PDMS preparations. These differences were due to adsorption of the head and/or neck to hydrophilic substrates. Because of the elasticity of PDMS, we propose that a PDMS membrane should be used for CASA. To investigate the dynamics of motile porcine sperms with microfluidics, we do not recommend plasma treatment to bond PDMS and glass in the microchannel preparation; instead, we suggest that a PDMS molding process without plasma treatment be used for preparation of microfluidic channels

Hiroaki FUNAHASHI

Keisuke YAMASHITA

Koji MATSUURA

TAKAYAMA SHUICHI

Yuka KURODA

Journal of Reproduction and Development

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Hydrophobic Silicone Elastomer Chamber for Recording Trajectories of Motile Porcine Sperms without Adsorption

Matsuura, Koji

Kuroda, Yuka

Yamashita, Keisuke

Funahashi, Hiroaki

Okayama University Scientific Achievement Repository

1  Hydrophobic silicone elastomer chamber for recording 1 trajectories of porcine motile sperms without adsorption 2  3 Koji MATSUURA1,*, Yuka KURODA1, Keisuke YAMASHITA2, Hiroaki FUNAHASHI2, 3 4  5 1Research Core for Interdisciplinary Sciences, Okayama University, Okayama, Japan 6 2Department of Animal Science, Faculty of Agriculture, Okayama University, Okayama, Japan 7 3Department of Animal Science, Graduate School of Natural Sciences and Technologies, 8 Okayama University, Okayama, Japan 9  10 Running head: Porcine Motile Sperms without Adsorption 11  12 *Corresponding author 13 E-mail address: kojimatu@md.okayama-u.ac.jp 14 Fax: +81-86-251-8456 15  16 Acknowledgments: This study was partly supported by a grant-in-aid for Scientific Research 17 for Young Scientists (B) (No. 20700380 to KM) by the Adaptable and Seamless Technology 18 Transfer Program through Target-Driven R&D from Japan Science and Technology Agency 19 2  (KM and HF) and by Special Coordination Funds for Promoting Sciences and Technology from 20 the Ministry of Education, Science, Sports, and Culture, Japan (KM).  21 22 3  Abstract  23 Porcine motile sperms adhere to hydrophilic materials such as glass and plastics. The 24 adsorption of sperms to a hydrophobic polydimethylsiloxane (PDMS) membrane was less 25 compared with that to glass. Significant decreases in linear velocity and amplitude of lateral head 26 displacement of motile porcine sperms were due to adsorption of the head and/or neck to the 27 hydrophilic substrates. Because of the elasticity of PDMS, we propose that a PDMS membrane 28 should be used for conventional Computer Assisted (Aided) Sperm Analysis. To investigate 29 dynamics of motile porcine sperms with microfluidics, we do not recommend plasma treatment 30 to bond PDMS and glass in the microchannel preparation; instead, we suggest that a PDMS 31 molding process without plasma treatment be used for preparation of microfluidic channels.  32   33 Key Words: porcine sperm motility, silicone elastomer, adsorption, trajectories 34 35 4  Introduction 36 Sperm motility analysis is a representative method for evaluation of male fertility, since 37 motility is correlated with viability [1-4]. The conventional method commonly referred to as 38 Computer Assisted (Aided) Sperm Analysis to record motility and linear velocity (LV) utilizing 39 a microscope with a charge coupled device [1-3]. The advantage of CASA over manual 40 observation is the absence of subjective calibration [1]. It is difficult to record trajectories of 41 motile porcine sperms and investigate LV related to fertility because they often adsorb to glass 42 and plastic which remarkably decreases their motility. To record trajectories of motile sperms 43 and investigate their velocity distribution quantitatively under a microscope, the use of 44 transparent materials that do not promote adsorption of motile sperms is necessary. 45 For observation of motile sperms, diluted semen is usually sandwiched between 46 hydrophilic glass slides [5]. Trajectories of human and bull sperms can be recorded using this 47 glass preparation; however, it is difficult to record the trajectory of motile porcine sperms, 48 because they adsorb to glass and hydrophilic plastics such as poly(methyl methacrylate) 49 (PMMA). We hypothesized that it may be possible to record the trajectory of these sperms using 50 transparent materials with high hydrophobicity represented by a high contact angle (>90 degrees) 51 to water droplets (Figure 1).  52 Hydrophobic silicone elastomer polydimethylsiloxane (PDMS), used at a contact angle of 53 110 degrees, is a key material capable of extending device applications for reproductive 54 technology because it is nontoxic, transparent, inexpensive, and easy to handle [6-11]. PDMS 55 microfluidic devices prepared by molding the microstructure and bonding the cured structure 56 with a cover or slide glass can be used for manipulation and culture of cells to investigate their 57 physiological functions [6-8]. Microfluidic channels are used for in vitro fertilization in case of 58 5  low sperm number (>105 cells) and for in vitro culture to mimic the oviduct environment [6, 9, 59 10]. Lopez-Garcia et al. observed bull sperm motions without adsorption to glass substrates in 60 glass-bottom PDMS microchannels [11]. Despite previous documented applications, there are 61 few practical applications for PDMS membranes combined with CASA in routine analysis. 62 In this study, using a PDMS preparation, we could record the trajectories of motile sperms 63 without adsorption and compare the sperm motility parameters. Furthermore, we reported that to 64 observe motile porcine sperm dynamics using microfluidic channels, the PDMS chamber should 65 be prepared without oxygen (O2) plasma treatment. This technology can be applicable for 66 recording live imaging and mechanics of porcine motile sperms that adhere to hydrophilic 67 materials [12].  68  69 Materials and Methods 70 The diluted semen samples were transported to the laboratory within 2 h of collection at 71 26–32 C. Spermatozoa were diluted at a concentration of 1 × 108 cells/ml with modified Modena 72 solution containing 5 mM cysteine and 20% (v/v) boar seminal plasma. This preparation follows 73 that outlined in previous reports [12].  74  75 Preparation of the Silicone Elastomer Chamber  76  A PMMA mold was fabricated for the recording chamber using a conventional 77 mechanical microdrilling process (MDX-40; Roland, Osaka, Japan). PDMS slabs and 78 membranes with microstructures were prepared by casting prepolymer (TSE 3032; Momentive 79 Performance Materials, Tokyo, Japan) at a 1:10 curing agent-to-base ratio against positive relief 80 features [9, 13]. The prepolymer was cured at 70 C for 1 h. Cured PDMS has a highly cross-81 6  linked 3D structure. To investigate differences in hydrophilic materials, the PDMS surface was 82 treated with plasma cleaner (PDC-32G; Harrick Plasma Inc., Ithaca, NY, USA). PDMS 83 microchannels without O2 plasma treatment were prepared by 1-step curing. 84  85 Motile Sperm Trajectory Recording 86  Using a BM ×10 lens (Nikon Co Ltd., Tokyo Japan), sperm and particle motion were 87 tracked with a sperm motility analysis system (SMAS) (Kaga Electronics Co. Ltd., Tokyo Japan). 88 Frame rate of sperm tracking using SMAS was 60 per second.  89  90 Statistical Analysis 91 The Student's t-test was used to determine differences in LV and average amplitude of 92 lateral head displacement (ALHD) between groups. P < 0.05 was considered significant.  93  94 Results and Discussion 95 Comparison of Absorption of Porcine Motile Sperms to Several Materials 96  Almost all the sperms adsorbed to slide glass 15 min after preparation, while the number 97 of sperms adsorbed to the PDMS membrane decreased (Figure 2). We found that more than half 98 of the motile sperms adsorbed to the hydrophilic substrate treated with O2 plasma 10 min after 99 preparation. Adsorption properties of porcine sperms to transparent materials are summarized in 100 Table 1. We observed that the hydrophobicity of substrate materials is important for adsorption. 101 To prevent adherence, the preparation should be made such that the contact angle of the 102 materials with water is more than 80 degrees. 103   104 7  Performance of Optimized Chambers and Sperm Motility Parameters 105 We compared LV distribution of motile porcine sperms inside chambers to quantitatively 106 investigate motility changes in relation to adsorption to hydrophilic substrates. The average LVs 107 1 and 15 min after glass preparation and 15 min after PDMS preparation were 34.3, 7.9, and 33.3 108 (µm/second), respectively. There was no significant difference between the distribution 1 min 109 after glass preparation and 15 min after PDMS preparation (P > 0.05). The average amplitude of 110 ALHD 1 and 15 min after glass preparation and 15 min after PDMS preparation were 5.4, 2.1, 111 and 3.7 (µm), respectively (P < 0.05). We suggest that the significant decreases in LV and 112 ALHD were due to adsorption of the head and/or neck to the hydrophilic substrate (Figure 2 and 113 3). 114  115 PDMS Preparation for Sperm Motility Analysis 116  Figure 4 shows the PDMS preparation for conventional CASA. To prevent overlap of 117 motile sperm images, we designed the preparation to decrease focal depth. Semen was 118 sandwiched with 2 PDMS sheets (Figure 4A, B, and C). Due to the elastic property of PDMS, 119 the lower membrane was deflected by the weight of the semen. The flat surface of the upper 120 membrane was turned up and faced across it. The thickness of the semen was approximately 0.1 121 mm, and we confirmed no overlap of sperm images (Figure 4C). With this preparation, we were 122 able to record trajectories and analyze the distribution of sperm motility parameters.  123  124 Live Imaging Application in PDMS Microchannels 125   Microchannels for sperm motility analysis can be easily prepared by PDMS soft 126 lithography; however, there is a problem with microchannel preparation after O2 plasma 127 treatment since hydrophilicity of PDMS increases. We compared sperm adsorption to a PDMS 128 8  microchannel with a cover glass on the bottom bonded with O2 plasma treatment (Channel A) to 129 a PDMS microchannel without O2 plasma treatment (Channel B) (Figure 5A and B). After 130 washing with diluted water, the number of adhered porcine sperms on the bottom of channels A 131 and B were approximately 700 and 100 (number/mm2), respectively (Figure 5C and D). This 132 result is consistent with the LV distributions (Figure 3A). When preparing the microchannel, the 133 standard bonding method for PDMS and glass by O2 plasma or UV light cannot be used due to 134 increases in hydrophilicity of the materials [15]. 135  Microchannels are important in sperm motility analysis because they allow the 136 trajectories of bull and human motile sperms to be evaluated [11,16]. Interestingly, it has been 137 reported that bull sperms tend to preferentially swim along the walls and that this phenomena 138 occurs during flow and no flow [11]. Koyama et al. designed a microfluidic device for sperm 139 chemotaxis with 3 inlets and 3 outlets to make a gradient in the chemotaxis chamber [17]. The 140 PDMS substrate and glass coverplate were bonded by exposure to air plasma that would decrease 141 the hydrophobicity of PDMS; a treatment which would not be suitable for analysis of porcine 142 sperm chemotaxis. Our results suggest that a PDMS-bottom microchannel without hydrophilic 143 treatments, such as O2 and air plasma, can be used to investigate the chemotaxis and fluid 144 mechanics of porcine motile sperms.  145  In conclusion, porcine motile sperms adhere to hydrophilic materials such as glass and 146 PMMA. The adsorption of sperms to the hydrophobic PDMS membrane was lesser than that to 147 glass. Because of the elasticity of PDMS, we propose the use of this preparation for conventional 148 CASA to reduce overlap of motile sperm images, which are artifacts of CASA. Because of the 149 potential sperm adhesion, we do not recommend O2 plasma treatment for bonding PDMS and 150 glass during investigation of the dynamics and chemotaxis of motile porcine sperms using 151 9  microfluidics. We suggest that only a PDMS molding process is suitable for preparation of 152 microfluidic channels to be used with motile porcine sperms.  153 154 10   Table 1. Comparison of contact angle and adsorption of motile sperms to transparent materials 155  Contact angle of water (deg) Adsorption References Glass 30 Yes 5 PMMA 70 Yes 14 PDMS 110 No 15 PDMS after O2 plasma treatment 50 Yes 15   156 157 11   FIGURE CAPTIONS  158 Figure 1. Definition of contact angles.  159  160 Figure 2. Differences in trajectories of fresh porcine sperms on (A) glass, (B) glass after 15 min, 161 and (C) PDMS membrane after 15 min.  162  163 Figure 3. (A) LV and (B) ALH distributions recorded on glass and PDMS preparations. 164  165 Figure 4. PDMS chambers (A) membrane for preparation having an area of 0.5 × 1 mm2, (B) 166 the method to sandwich semen between the 2 membranes, (C) cross-sectional image for 167 recording the trajectories of motile sperms. Dark and light gray objects represent the PDMS 168 membrane and semen, respectively. (D) Sperms in this preparation in CASA. 169  170 Figure 5. (A) Live imaging of porcine motile sperms in PDMS microchannels. 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Hydrophobic Silicone Elastomer Chamber for Recording Trajectories of Motile Porcine Sperms without Adsorption

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