Accepted Manuscript

Improved bovine in vitro embryo production with sexed and unsexed sperm selected
by chemotaxis

Esteban Mauricio Dominguez, Ayelen Moreno-Irusta, Héctor Alejandro Guidobaldi,

Huberto Tribulo, Laura Cecilia Giojalas

PII: S0093-691X(18)30720-9
DOI: 10.1016/j.theriogenology.2018.08.023
Reference: THE 14682

To appear in: Theriogenology

Received Date: 21 February 2018

Revised Date: 24 August 2018
Accepted Date: 26 August 2018

Please cite this article as: Dominguez EM, Moreno-Irusta A, Guidobaldi HéAlejandro, Tribulo H, Giojalas
LC, Improved bovine in vitro embryo production with sexed and unsexed sperm selected by chemotaxis,
Theriogenology (2018), doi: 10.1016/j.theriogenology.2018.08.023.

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 ACCEPTED MANUSCRIPT

1 "Revised"

2 Improved bovine in vitro embryo production with sexed and unsexed sperm

3 selected by chemotaxis

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5 Esteban Mauricio Dominguez1, 2, Ayelen Moreno-Irusta 1,2, Héctor Alejandro

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6 Guidobaldi1, 2, Huberto Tribulo 3 and Laura Cecilia Giojalas1, 2,*

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8 Universidad Nacional de Córdoba (UNC), Facultad de Ciencias Exactas, Físicas

9 y Naturales, Centro de Biología Celular y Molecular, Córdoba, CP 5016, Argentina

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2

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Consejo de Investigaciones Científicas y Técnicas (CONICET), Instituto de
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11 Investigaciones Biológicas y Tecnológicas, Córdoba, CP 5016, Argentina.
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12 Instituto de Reproducción Animal Córdoba, Estación Gral. Paz, Paraje Pozo del
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13 Tigre, CP 5145, Córdoba, Argentina.

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15 Corresponding author

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25 Abstract

26 Assisted reproductive techniques (ART) have been widely used in farm animals in

27 the last decades. Sexed cryopreserved spermatozoa, ovum pick up, in vitro

28 embryo production and transfer constitute the ART that have revolutionized the

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29 dairy industry. However, the efficiency of some of these techniques is still low due

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30 in part to sperm quality, which influences fertilization, embryo development and

31 implantation. The Sperm Selection Assay (SSA), based on sperm chemotaxis

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32 towards progesterone, provides a sperm subpopulation enriched with spermatozoa

33 that are capacitated, with intact DNA and low level of oxidative stress. Since the

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SSA selects a sperm subpopulation at optimum physiological state, the application
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35 of the SSA may improve the efficiency of the current ART. The aim of this study

36 was to adapt the SSA for unsexed and sexed bovine frozen-thawed semen
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37 samples, and then to test whether sperm selection by the SSA improves the
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38 cleavage rate of bovine embryos in vitro. The optimal SSA conditions to obtain the
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39 higher sperm accumulation percentage given by chemotaxis were the same for

40 both unsexed and sexed semen samples. Thus, sperm accumulation in W2 was
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41 significantly higher when: 2 million sperm per mL were placed in W1 (unsexed

42 samples: 12 ± 1%, p = 0.002; sexed samples: 14 ± 3%, p = 0.02); 1 pM

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43 progesterone was placed in W2 (unsexed sample: 9 ± 1%, p = 0.009; sexed

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44 samples: 11 ± 2%, p = 0.02); and to incubate the SSA device for 10 minutes

45 (unsexed samples: 17 ± 2%, p = 0.007; sexed samples: 10 ± 1%, p = 0.004). We

46 found that the quality of spermatozoa recovered from W2 in unsexed and sexed

47 semen was enhanced. Thus, the capacitation index was significantly increased

48 (unsexed samples: 1.75 ± 0.1, p =0.0001; sexed samples: 1.76 ± 0.2, p = 0.004),
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49 while DNA fragmentation index was significantly decreased (unsexed samples:

50 0.33 ± 0.07, p = 0.0003; sexed samples: 0.32 ± 0.04, p = 0.002). Moreover, the

51 cleavage index of oocytes fertilized with either unsexed or sexed SSA-selected

52 sperm was significantly improved (unsexed samples: 3.2 ± 0.4, p = 0.0001; sexed

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53 samples: 2.3 ± 0.33, p = 0.03). Thus, we show that the SSA can be used to recruit

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54 a bovine sperm subpopulation at optimal functional state regardless of whether the

55 sample is previously sexed, and that this optimal state improves bovine embryo

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56 cleavage rate.

57 Keywords

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Bovine sperm; sperm chemotaxis; sperm capacitation; sperm DNA fragmentation;
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59 in vitro fertilization.

60
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61 1. Introduction
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62 In the last decades, the application of ART has notably improved animal
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63 production, but these techniques are still far from being efficient and low cost. The

64 dairy industry is a competitive market where animals with the highest genetic
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65 potential are continuously selected for ART. Oocyte recovery is optimized either by

66 ovum pick up [1] from pregnant cows until 3 to 4 gestation months [2], or by
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67 laparoscopic ovum pick up from 2 to 3 months old prepubescent calves [3]. These
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68 enhanced oocyte technologies are used to produce embryos in vitro, which are

69 later transferred to receptive cows. These techniques, combined with sexed

70 spermatozoa samples, are preferred to obtain an offspring of female embryos,

71 which are in high demand in the dairy industry.

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72 Despite the potential benefits of the application of sexed semen technology

73 in bovine, the percentage of embryos obtained with sexed spermatozoa is lower

74 than the one obtained with unsexed spermatozoa [4]. Moreover, the pregnancy

75 rate of cows artificially inseminated with sexed semen is lower than the rate

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76 obtained with unsexed spermatozoa [5-7]. These data suggest that the

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77 spermatozoa sexing process may alter sperm quality by inducing DNA damage

78 due to the use of fluorescent dyes and UV light stimulation during sperm sorting [8-

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79 10]. Recent literature reported that sexed spermatozoa artificially inseminated lead

80 to a reduction of in vivo fertility, but the blastocyst rate is similar to that obtained

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with unsexed semen under in vitro fertilization [11]. In any event, it would be
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82 advantageous if optimally functioning spermatozoa could be separated from those

83 with damaged DNA before using them for ART.

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84 Recently, a sperm selection assay (SSA) has been developed in our

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85 laboratory. This technique enables the separation of a sperm subpopulation

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86 enriched in fertilizing spermatozoa (called `capacitated sperm´), which present low

87 DNA fragmentation and oxidative stress [12]. Thus, the SSA facilitates the
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88 recruitment of physiologically optimal spermatozoa via chemotaxis (a cell chemical

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89 guidance) [13,14] towards very low doses of progesterone (P; picomolar range), a
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90 hormone secreted by the cumulus cells by the time of ovulation [15–19]. Such

91 selected sperm population at optimum physiological state might support not only

92 fertilization but also early embryo development and implantation [20,21]. Since the

93 spermatozoa selected by SSA present intact DNA, the application of this assay to

94 bovine samples will result in physiologically competent unsexed and sexed

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95 samples. The aim of this study was to adapt the SSA, originally developed for

96 human spermatozoa, for unsexed and sexed bovine semen samples, and to test

97 whether by selecting unsexed or sexed spermatozoa by chemotaxis the cleavage

98 rate is improved in bovine embryos in vitro.

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99 Here we show that both unsexed and sexed frozen-thawed sperm

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100 subpopulations selected by chemotaxis by the SSA are enriched in capacitated

101 spermatozoa which present lower DNA fragmentation, and that the use of these

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102 selected spermatozoa in in vitro fertilization improves bovine embryo cleavage

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103 rate. AN
104 2. Materials and Methods

105 2.1 Reagents

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106 All chemicals were purchased from Sigma-Aldrich (St. Lois, MO, USA),

107 unless otherwise indicated.

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108 2.2 Sperm preparation

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109 Experiments were carried out with cryopreserved bovine semen samples.

110 The unsexed samples were provided by Instituto de Reproducción Animal de

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111 Córdoba (Córdoba, Argentina), and the sexed samples were purchased from
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112 GENPRO (Santa Fe, Argentina). Frozen unsexed samples were obtained from two

Holando Argentino and two Braford bulls (20x106 sperm/mL, 0.5 mL per straw).
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114 Frozen sexed samples were obtained from two Holando Argentino bulls (2x106

115 sperm/mL, 0.25 mL per straw). Thus, replicates in each set of experiments were

116 performed with different ejaculates from different bulls. On the day of the

117 experiment, semen samples were thawed at 37ºC for 20-30 seconds. Then, the
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118 seminal plasma and cryoprotectant in unsexed samples were removed by the

119 migration-sedimentation technique [22], and by simple centrifugation in sexed

120 samples [23]. Spermatozoa were then diluted in Sp-TALP medium [24],

121 supplemented with 3 mM pentoxifylline [25,26], and further incubated for 1 hour at

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122 38.5°C, in an atmosphere of 5% CO2 in air. The percentage of motile spermatozoa

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123 was determined at the end of the incubation time by video microscopy and image

124 analysis [27] with a phase contrast microscope (Nikon Instruments Inc., NY,USA)

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125 and NIS elements software (4.30.01 DU1). The movement of spermatozoa was

126 digitally recorded for 10 s at 30 Hz at 10x, and videos were analyzed by FIJI

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software using the plug-in motility tool. In every experiment, only samples
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128 exhibiting ≥90% of motile spermatozoa were used.

129 2.3 Sperm Selection Assay

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130 The SSA was performed according to Gatica et al [11]. Briefly, the device
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131 used in the SSA consists of two wells; the sperm suspension is placed in one well
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132 (W1), and the attractant solution (or culture medium as negative control) is placed

133 in the other (W2). The two wells are connected by a tube, in which a gradient is
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134 formed as the attractant solution diffuses from W2 to W1, thus inducing sperm

135 chemotaxis. Either bovine follicular fluid (FF) or P were used as attractant [28,29]
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136 as described in the results section. After loading, the device was incubated at
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137 38.5°C, in an atmosphere of 5% CO2 for different time periods, as specified in the

138 results section. At the end of the assay, the net sperm accumulation in W2 was

139 determined. This parameter was calculated as the difference in the percentage of

140 spermatozoa recovered from W2 with or without attractant solutions.

141 2.4 Sperm capacitation

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142 The ability of spermatozoa to undergo the induced acrosome reaction was

143 determined as an indirect indicator of the level of capacitated spermatozoa

144 [27].The acrosome status was detected by in vivo Pisum sativum agglutinin

145 fluorescein staining (PSA-FITC) [30,31], whereas a similar procedure was also

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146 employed in bull spermatozoa samples [32]. Briefly, the sperm suspension was

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147 divided into two aliquots, which were incubated with or without 8 µM of the calcium

148 ionophore A23187 and 10 µg/mL PSA-FITC in culture medium, at 38.5°C for 30

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149 min. Then, the sperm suspensions were fixed in 2% formaldehyde in PBS for 20

150 min at room temperature, washed by centrifugation in distilled water, and the pellet

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was let to air dry on a slide. The status of the acrosome was observed at 1,000x
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152 under a fluorescence microscope (Olympus BX 50, Center Valley, USA). Under the

153 microscope, the acrosome-reacted spermatozoa present a green fluorescent

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154 acrosome, while the ones with intact acrosome are unlabeled. The percentage of
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155 capacitated spermatozoa was determined as the percentage difference between

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156 induced and spontaneous acrosome-reacted spermatozoa (n=200 cells). A

157 capacitation index was determined as the ratio of the percentage of capacitated
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158 spermatozoa obtained before and after the SSA, with or without progesterone, to

159 the percentage of capacitated spermatozoa before the SSA (control without sperm
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160 selection).
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161 2.5 Sperm DNA fragmentation

162 The level of DNA fragmentation was evaluated by the Sperm Chromatin

163 Dispersion assay (SCD), as described by Fernandez et al [33], with minor

164 modifications [34]. The sperm suspension was mixed with 1% low-melting point

165 aqueous agarose at 37°C, and then 50 µl aliquots were placed on a slide pre-
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166 coated with 0.65% standard agarose and dried at 80°C. The sample was covered

167 with a coverslip and let solidify at 48°C for 30 minutes. Then, coverslips were

168 carefully removed, and slides were immediately immersed horizontally in a tray

169 with freshly prepared acid denaturation solution (0.08 N HCl) at 22°C for 7 min, in

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170 the dark. The slides were incubated in a neutralizing and lysis solution 1 (0.4 M

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171 Tris, 5% 2-Mercaptoethanol, 1% SDS, 50 mM EDTA, pH 7.5) for 10 min at room

172 temperature, in order to stop DNA denaturation and for protein removal, followed

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173 by incubation in neutralizing and lysis solution 2 (0.4 M Tris, 2 M NaCl, 1% SDS,

174 pH 7.5) for 5 min at room temperature. Slides were thoroughly washed in Tris

175
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borate-EDTA buffer (0.09 M Tris-borate,0.002 M EDTA, pH 7.5) for 2 min, and
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176 dehydrated in sequential ethanol series. Dried sperm samples were stained with

177 Hoechst 33258 (1 µg/mL). Images of sperm heads from at least 200 cells per
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178 treatment were obtained with a fluorescence microscope (Olympus BX 50,

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179 Olympus, Center Valley, USA) coupled to a Nikon digital camera (Nikon
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180 Instruments Inc., NY, USA). The halo area observed around the head of each

181 spermatozoon was measured using the FIJI program. Sperm heads were classified
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182 according to the size of the halo (µm2). Four halo patterns were identified: large,

183 60±10 µm2; medium, 30±10 µm2; small, 10±10 µm2, and without halo. The first two
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184 patterns include sperm without DNA fragmentation, while the other two patterns
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185 include fragmented DNA cells. The DNA fragmentation index was determined as

186 the ratio of the percentage of fragmented DNA spermatozoa obtained before and

187 after the SSA, with or without P, to the percentage of fragmented DNA

188 spermatozoa before the SSA (control without sperm selection).

189 2.6 Oocyte collection and in vitro maturation

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190 Ovaries from mature cows were obtained from an abattoir (Bustos y Beltran

191 SA, Córdoba, Argentina), preserved at 22–25°C in a 0.9% NaCl solution

192 supplemented with 100 IU/mL penicillin and 100 mg/mL streptomycin sulfate, and

193 transported to the laboratory within 6 h. The ovaries were cleaned from

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194 surrounding tissue, and rinsed in 0.9% NaCl. Follicles under 13 mm size were

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195 aspirated with a 19G needle. Grade I, II and III COCs (cumulus-oocyte complexes)

196 [35] were selected under a stereoscopic magnifying glass for in vitro maturation,

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197 rinsed three times in holding medium consisting of TCM 199 Hank´s Salts (Gibco

198 Life Technologies, USA), supplemented with 10% fetal calf serum (FCS), 0.3 % faf-

199
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BSA, and 0.05 mg/mL gentamycin. Then, the COCs were let matured in maturation
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200 medium consisting of TCM 199 Earle’s Salts (Gibco Life Technologies, USA), plus

201 1% FCS, 0.01 UI/mL rhFSH (Gonal F, Merck Laboratory, USA), 0.1 mg/mL
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202 glutamine, 2.5 mg/mL sodium pyruvate, and 0.05 mg/mL µg gentamycin. Twenty
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203 COCs per 100 µl maturation medium droplets were incubated 22 to 24 h at 38.5°C
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204 in an atmosphere containing 5% CO2 covered with mineral oil, in 60x15 mm Petri

205 dishes.
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206 2.7 In vitro fertilization and embryo culture

207 At the end of the maturation period, COCs were rinsed three times with IVF-
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208 SOF medium (315 mg NaCl, 26.8 mg KCl, 2 mg KH2PO4, 105 mg NaHCO3, 3.25
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209 mg Penicillin, 2.75 mg C3H3O3Na, 400 mg faf-BSA, 4.5 mg C6H12O6, 12.6 mg

210 CaCl2H2O, 23.5 µl C3H5O3Na [60% Syrup], 50 µg/mL heparin, in 50 mL of ultra-

211 pure water). Then, five matured oocytes were placed in a 30 µl IVF-SOF medium

212 droplet and covered with mineral oil, in 60x15 mm Petri dishes. Ten microliters of

213 the sperm suspension were added to the droplet containing the COCs to get 1,000
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214 spermatozoa per oocyte, according to the rate at which the percentage of cleavage

215 obtained is approximately 20%, as described for unsexed and sexed samples by

216 Trigal et al [36] . This experimental strategy allows to determine the magnitude of

217 the increment in the embryo cleavage index upon sperm selection by the SSA. The

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218 gamete co-incubation time point was considered as day zero of fertilization.

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219 Gametes were co-incubated 16 to 18 h at 39°C in a high humidity atmosphere

220 enriched with 5% CO2. Presumptive zygotes were vortexed for 2 min to separate

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221 cumulus cells, and washed three times in SOF-Citrate medium (314.5 mg NaCl,

222 26.5 mg KCl, 8.1 mg KH2PO4, 9.1 mg MgSO4.7H2O, 105 mg NaHCO3, 2.5 mg

223
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Gentamicin, 4 mg C3H3O3Na, 150 mg faf-BSA, 1.5 mL BME, 0.5 mL MEM, 2.5 mg
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224 C5H10N2O3, 60 mg acid free HEPES, 5 mg C6H5Na3O7, 25 mg Myo–inositol, 65 mg

225 sodium salt HEPES, 13.1 mg CaCl2.H2O, 15 µl C3H5O3Na [60% Syrup] in 50 mL of

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226 ultra-pure water). Twenty zygotes per 100 µl SOF-citrate media droplets were
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227 incubated covered with mineral oil at 38.5°C in an atmosphere containing 5% CO2
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228 and 5% O2 in air with high humidity.

229 2.8 Embryo cleavage

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230 At day 2, the occurrence of cleavage, indicated by the presence of two or more

231 cells, was verified under inverted phase contrast microscopy (Olympus CK 2,
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232 Japan) at 200x magnification. The percentage of embryo cleavage was calculated
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233 as the ratio of the number of zygotes undergoing mitotic division to the total

234 number of mature oocytes incubated with spermatozoa. To know how many times

235 the cleavage rate was modified by fertilization of oocytes with SSA-selected

236 spermatozoa , the cleavage rate index was calculated as the rate of the

237 percentage of cleaved embryos obtained with spermatozoa selected by the SSA
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238 (with or without P) to the percentage of embryos obtained with spermatozoa not

239 selected by the SSA. An embryo parthenogenetic control was performed in every

240 experiment, and parthenogenic division was not observed.

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241 2.9 Statistical analysis

242 Differences between treatments were determined by means of a two-way

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243 ANOVA, and a posteriori Tukey test performed with the Graph Pad Prism 5.01 (La

244 Jolla, CA, USA). The parameters expressed as percentages were previously

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245 transformed to the arcsine square root of the proportion. To evaluate the

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246 correlation between the percentage of cleaved embryos and the percentage of
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247 sperm capacitation or DNA fragmentation, Pearson’s correlation coefficient

248 analyses were performed. P values less than 0.05 were considered statistically
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249 significant.

250 3. Results
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251 The SSA experimental conditions were adapted for frozen-thawed unsexed

252 and sexed bovine sperm samples. To define the optimum timing and sperm
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253 concentration to run the SSA, 10-3 FF (a known chemoattractant for bovine

254 spermatozoa; [29]) was placed in W2, whereas the chemotactic activity of the
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255 batch was previously determined (Suppl 1). Firstly, 2 million sperm per mL were
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256 placed in W1 and 10-3 FF in W2, incubating the SSA under different incubation

257 times (5, 10 and 20 min; Fig. 1Ai). A 10 min incubation was enough to get a

258 significant sperm accumulation (unsexed: p = 0.007, sexed: p = 0.004; Fig. 2A).

259 Then, different sperm concentrations were placed in W1 (1, 2 and 3 million sperm

260 per mL) and 10-3 FF in W2, incubating the SSA device for 10 min (Fig. 1Aii). When
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261 2 million sperm per mL were placed in W1 a significant sperm accumulation was

262 observed in W2 (unsexed: p = 0.002, sexed: p = 0.02; Fig. 2B). Under these

263 experimental conditions, we next performed a dose response experiment to verify

264 whether unsexed and sexed bovine spermatozoa also chemotactically orient their

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265 movement towards a concentration gradient of P (Fig.1Aiii). When 1 pM P was

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266 placed in W2, a significant increase in the level of sperm accumulation in W2 was

267 observed (unsexed: p = 0.009, sexed: p = 0.02; Fig. 2C). No statistically significant

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268 differences were observed between unsexed and sexed samples for each set of

269 experiments (p = 0.08, p = 0.6, p = 0.09, corresponding to Fig. 2A, B and C,

270 respectively).
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271 Sperm chemotaxis towards 1 pM P was further verified in unsexed and

272 sexed semen samples by performing several controls of the SSA, loading the
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273 hormone in different combinations (Fig. 1Bi-iii). Since chemotaxis is dependent on

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274 the gradual distribution of the attractant molecule, when P was homogeneously
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275 distributed along the SSA device no gradient formed between wells, no sperm

276 accumulation was observed. Conversely, when the attractant was placed together
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277 with spermatozoa in W1, the cells sensed a descending gradient of P (from W1 to

278 W2), and consequently, they came back to W1 and accumulate there, causing a
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279 significant depletion of spermatozoa in W2 (unsexed: p = 0.0001, sexed: p =

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280 0.0006; Fig. 2D). A further rationale for these experimental controls can be seen in

281 Giojalas et al [13]. No statistically significant differences were observed between

282 unsexed and sexed samples (p = 0.3). In summary, the optimum conditions to

283 perform the SSA with frozen-thawed unsexed and sexed sperm samples are to
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284 place 2 million sperm per mL in W1 and 1 pM P in W2, incubating the SSA device

285 for 10 min, conditions that were applied in the following experiments.

286 Next, we verified the efficiency of the SSA to recruit the competent unsexed

287 and sexed bovine spermatozoa. After incubating spermatozoa under capacitating

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288 conditions, the samples were divided in three groups: sperm not selected by SSA

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289 (“Before SSA”), SSA-selected sperm in the presence of P (after SSA +P), and

290 SSA-selected sperm in absence of P (after SSA –P), and the indexes of sperm

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291 capacitation and DNA fragmentation were determined (Fig. 1C). We found that the

292 index of capacitated spermatozoa recovered from W2 in the presence of P was

293
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significantly higher than the index without P, or before SSA, in both unsexed and
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294 sexed samples (unsexed: p = 0.0001, sexed p = 0.004; Fig. 3A). Moreover, the

295 level of spontaneous acrosome reaction was low enough to visualize the significant
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296 increment in induced AR caused by A23187 and did not differ between treatments
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297 (Suppl table. 1). In addition, the DNA fragmentation index was significantly lower
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298 after the SSA in the presence of P (unsexed: p = 0.0003, sexed: p = 0.002; Fig.3

299 B). No statistically significant differences were observed between unsexed and
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300 sexed samples (p = 0.69 and p = 0.76, respectively).

301 Then, unsexed and sexed spermatozoa before SSA or recruited with or
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302 without P in the SSA device, were used to fertilize oocytes under in vitro
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303 conditions, determining the cleavage index (Fig. 1C). When the oocytes were

304 fertilized in vitro with spermatozoa chemotactically selected by P in the SSA, the

305 cleavage index was significantly improved compared to those oocytes fertilized

306 with spermatozoa before the SSA or those used in the SSA without P (unsexed: p

307 = 0.0001, sexed: p = 0.03; Fig. 3 C). No statistically significant differences were
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308 observed between unsexed and sexed samples (p = 0.49). The total number of

309 oocytes and cleaved embryos per treatment are shown in Table 1. The percentage

310 of sperm capacitation and DNA fragmentation determined in the population before

311 and after the SSA, with and without P, was correlated with the percentage of

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312 cleaved embryos, for unsexed and sexed sperm samples. The level of sperm

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313 capacitation was positively and significantly correlated with the embryo cleavage

314 rate, in frozen-thawed unsexed (Fig. 4 A) and sexed (Fig. 4 B) spermatozoa. In

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315 other words, the higher the level of capacitated sperm, the higher the embryo

316 cleavage rate. The level of sperm DNA fragmentation was inversely correlated to

317
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the embryo cleavage rate in frozen-thawed unsexed (Fig. 4 C) and sexed (Fig. 4 D)
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318 spermatozoa; thus, the lower the level of fragmented DNA spermatozoa, the higher

319 the embryo cleavage rate. The respective correlation coefficients were very similar
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320 between unsexed and sexed samples as shown in the respective figures.
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321
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322 4. Discussion

323 We have previously described the in vitro conditions in which the Sperm
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324 Selection Assay (SSA) is applied to human sperm samples in order to enrich the

325 capacitated sperm population in a given sample [12]. Here, we show that the SSA
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326 can be easily adapted to be used with frozen-thawed, unsexed and sexed bovine
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327 spermatozoa, and that the selected spermatozoa present an optimum physiological

328 state that enhances the embryo cleavage rate.

329 Defining the SSA experimental conditions is critical, since the efficiency of the

330 assay is strictly dependent on assay time and on the sperm and attractant

331 concentrations [12]. When applied to fresh human, rabbit and mouse spermatozoa,
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332 the SSA device is incubated for 20 min in the presence of the same picomolar

333 progesterone concentration added to W2, while the sperm concentration loaded in

334 W1 varies according to the species [37]. Here we used frozen-thawed

335 spermatozoa, which may differently respond to the SSA, hence we first adjusted

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336 the experimental conditions to this species, which indeed, were slightly different

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337 from those observed in others. For instance, the optimum sperm concentration and

338 the duration of the SSA are shorter than those set up for other species

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339 spermatozoa. This may be due to the addition of pentoxifylline, which stimulates

340 sperm motility, and so more mobile spermatozoa can rapidly accumulate in the

341
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SSA well containing P. At the beginning of the study, we did not know that bovine
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342 spermatozoa could also be attracted by progesterone. Therefore, the first setting of

343 the SSA was done with FF, a known attractant for spermatozoa of several species
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344 including bulls [29]. Once the basic conditions were set up with FF, we tested the
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345 sperm chemotactic attraction towards P. Although the optimum P concentration to

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346 perform the SSA is one order of magnitude lower than that concentration

347 necessary to recruit spermatozoa from other species, the chemotactic response is
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348 still displayed at the picomolar range [28]. Moreover, according to the SSA

349 controls, the sperm recruited in W2 is only mediated by chemotaxis as also

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350 observed in other species [12,38]. This result validates the elemental virtue of
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351 chemotaxis, its ability to select capacitated spermatozoa.

352 Gamete cryopreservation alters some cellular functions, mainly its DNA

353 integrity [38]. Even though the in vitro performance of fresh semen samples is

354 higher than that of cryopreserved samples [39], ART procedures are also

355 performed with frozen-thawed sperm samples due to the need for gamete
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356 manipulation and transportation [40]. Here we showed that the outcome of the SSA

357 performed with bovine frozen-thawed samples was similar to that obtained with

358 fresh human semen samples (around 10%, expected value of capacitated-

359 chemotactic spermatozoa at any given time) [11]. Therefore, sperm damage

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360 caused by cryopreservation procedures may be minimized by performing the SSA

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361 after thawing, probably due to its ability to recruit those cells that are in good

362 physiological condition, as observed in frozen-thawed unsexed and sexed semen

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363 samples.

364 For economic reasons, sex-selected spermatozoa combined with ART are

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applied to spermatozoa from several species; for instance, to replace dairy cattle or
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366 to generate polo mare offspring. The most accepted sperm sexing method is cell

367 sorting. Since this technique involves nuclear staining and laser illumination,
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368 several undesired effects may be observed in sexed sperm samples, such as
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369 reduced sperm viability and DNA damage [41]. In this context, the ability of the
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370 SSA to sort out DNA-damaged spermatozoa and to recruit capacitated

371 spermatozoa can be appreciated equally in unsexed and sexed semen samples.
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372 Even though differences in number of bulls, straw volume and thawing

373 timing of sexed samples might differentially affect the SSA results as compare to
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374 unsexed samples, if this were the case, the expected results would be a poor
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375 response in sexed samples due to the mentioned factors. Nevertheless, this was

376 not the case since no significant differences were found between unsexed and

377 sexed samples.

378 It has been shown that embryos produced by in vitro oocyte fertilization with

379 bovine sexed spermatozoa, present reduced expression of genes involved in

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380 development [4]. In addition, these authors observed that the embryo cleavage rate

381 was around 30% below those obtained with unsexed spermatozoa. Here we

382 showed that oocytes fertilized in vitro with sexed spermatozoa previously selected

383 by the SSA, showed similar rates of embryo cleavage to those fertilized with

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384 unsexed spermatozoa. Moreover, the oocytes fertilized in vitro with unsexed SSA

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385 selected spermatozoa also provided an enhanced cleavage rate. Interestingly, we

386 found that sperm capacitation and low DNA fragmentation are significantly

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387 correlated with embryo cleavage rate, which emphasizes the importance of sperm

388 physiological status. Thus, the SSA biotechnology could be beneficial not only for

389
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dairy cattle production but also for the reproduction of other species where the
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390 female embryos are also preferred.

391
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392 5. Conclusion
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393 Here we show that the use of the SSA with frozen-thawed bovine spermatozoa
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394 is as efficient as with human sperm samples. We found that the quality of the

395 sexed sperm population recovered from SSA in the presence of progesterone is
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396 similar to the quality of the unsexed population. Finally, we determined that

397 fertilization of oocytes by unsexed or sexed spermatozoa selected by SSA in the

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398 presence of progesterone results in a higher embryo cleavage rate.

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399

400 Conflict of interest

401 CONICET and UNC are the owners of the SSA patent. L.C.G and H.A.G are

402 inventors of the SSA device.

403
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404 Acknowledgments

405 L.C.G. and H.A.G. are Principal Investigator and Associate Investigator from

406 CONICET, respectively. E.M.D. and A.M.I are PhD students from Universidad

407 Nacional de Río Cuarto (Argentina) and Universidad Nacional de Córdoba,

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408 respectively, and CONICET fellowship holders. The authors thank the advice of Dr.

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409 Francisco Ludueña, statistician of our institute.

410

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411 Authors’ contribution

412 E.M.D conceived and designed the experiments, acquired, analyzed, and

413
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interpreted data; wrote and revised the article. A.M.I acquired, analyzed and
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414 interpreted data. H.A.G interpreted data and revised the article. H.T revised the

415 article. L.C.G conceived and designed the experiments; interpreted data, wrote the
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416 article, and obtained funding.

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417
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418 Funding

419 The study received financial support from Agencia Nacional de Promoción
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420 Científica y Tecnológica (PICT Regional “A” – 1995 - 2011) and Universidad

421 Nacional de Córdoba (203/14; 313/16).

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422
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423 References

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536 doi:10.1016/S0093-691X(01)00494-0.
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540 4640.2003.tb02641.x.

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542 Neild DM. Evaluation of DNA fragmentation in llama (Lama glama) sperm
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543 using the sperm chromatin dispersion test. Anim Reprod Sci 2012;131:63–

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548 doi:http://dx.doi.org/10.1016/0093-691X(94)00020-U.

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552 doi:10.1016/j.theriogenology.2012.06.018.

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553 [37] Guidobaldi HA, Cubilla M, Moreno A, Molino M V, Bahamondes L, Giojalas

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555 fertilization. Hum Reprod 2017;32:1560–73.

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558 Implications for ART. Adv Urol 2012;2012:854837.

559 doi:10.1155/2012/854837.
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560 [39] Richter MA, Haning RV, Shapiro SS. Artificial donor insemination: fresh
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561 versus frozen semen; the patient as her own control. Fertil Steril
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562 1984;42:277–80.

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564 tissue or testicular cell suspensions : a pivotal step in fertility preservation.

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566 [41] Hamano K. Sex Preselection in Bovine by Separation of X- and Y-

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567 Chromosome Bearing Spermatozoa. J Reprod Dev 2007;53:27–38.

568 doi:10.1262/jrd.18141.

569

570 Legends for figures

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571 Figure 1. Adaptation of the experimental conditions to apply the SSA to

572 frozen-thawed bovine spermatozoa. Ai, 2 million sperm per mL were loaded in

573 well 1 (W1), and 10-3 FF were added to well 2 (W2), and the SSA device was

574 incubated for 5, 10, or 20 min; Aii, 1, 2, or 3 million spermatozoa per mL were

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575 placed in W1, and 10-3 FF in W2, and the SSA was incubated for 10 min; Aiii, 2

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576 million sperm per mL were placed in W1, and 0.1, 1, or 10 pM progesterone (P)

577 was added to W2 for 10 min. Bi, 2 million sperm per mL were placed in W1, and 1

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578 pM P in W2, generating an ascending P gradient from W1 to W2. Bii, 2 million

579 sperm per mL were loaded to W1, and 1 pM P were added to both wells, and thus

580
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it spread to the connecting tube between them, creating no attractant gradient; Biii,
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581 2 million sperm per mL and 1 pM P were placed in W1, while culture medium

582 without attractant was placed in W2, generating a descending gradient of P from
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583 W1 to W2. C, The indexes of sperm capacitation and DNA fragmentation were
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584 determined in the sperm population before performing the assay as well as in
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585 spermatozoa recovered from W2 after the SSA, with or without P. Embryo

586 cleavage induced in oocytes fertilized with spermatozoa from each of the three
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587 groups was also measured. Sp: spermatozoa, FF: follicular fluid, P: progesterone,

588 CM: culture medium.

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589
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590 Figure 2. Experimental conditions to apply the SSA with frozen-thawed

591 unsexed and sexed bovine semen samples. A, Percentage of sperm

592 accumulation in W2 as a function of SSA incubation time; B, sperm concentration

593 loaded in W1; C, progesterone concentration added to W2; and D, experimental

594 controls to verify sperm selection mediated by chemotaxis. Follicular fluid was used
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595 as the chemoattractant in experiments shown in A and B, while the other

596 experiments were performed with progesterone. White and black bars represent

597 unsexed and sexed sperm samples, respectively. Data are expressed as the

598 mean+SEM of independent experiments performed with different ejaculates from

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599 different bulls (three to five experiments were performed with unsexed samples,

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600 and four to six with sexed samples). Different letters indicate statistically significant

601 differences between treatments of the same sperm samples (sexed or unsexed).

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602

603 Figure 3. Application of the SSA to enhance embryo cleavage. A, Sperm

604
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capacitation index; B, DNA fragmentation index; C, embryo cleavage index, before
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605 and after the SSA performed with or without progesterone. White and black bars

606 represent unsexed and sexed sperm samples, respectively. The corresponding
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607 average percentage is shown above each bar. Data are expressed as the
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608 mean+SEM of independent experiments performed with different ejaculates from

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609 different bulls (seven performed with unsexed samples, and three with sexed

610 samples). Different letters indicate statistically significant differences between

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611 treatments of the same sperm samples (sexed or unsexed).

612
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613 Figure 4. Correlation between embryo cleavage and sperm parameters. A and
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614 B, percentage of sperm capacitation vs. percentage of embryo cleavage, for

615 unsexed and sexed samples, respectively. C and D, percentage of sperm DNA

616 fragmentation vs. percentage of cleavage, for unsexed and sexed samples,

617 respectively. Spermatozoa before (white dots) and after the SSA, either with the
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618 addition of progesterone (black dots) or without it (gray dots). The Pearson

619 correlation values (r) and p values are shown in each figure.

620

621 Supplementary Figure 1. Verification of the follicular fluid chemotactic

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622 activity. Unsexed 2 million spermatozoa per mL were loaded in W1 and several

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623 FF concentrations were added to W2, and percentage of sperm accumulation in

624 W2 was determined at the end of the assay. Data are expressed as the

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625 mean+SEM of four independent experiments, different letters indicate statistically

626 significant differences between follicular fluid concentrations.

627
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628

629
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630 Table 1. Number of total oocytes and cleaved embryos obtained per
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631 treatment and type of sperm sample.

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Sperm sample Treatment N° N° cleaved
633 oocytes embryos
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Before SSA 155 22

634 Unsexed After SSA without P 167 40
After SSA with P 176 70
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Before SSA 112 20
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Sexed After SSA without P 164 43

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After SSA with P 167 67
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637

638 Embryos were evaluated on day 2 (cleavage). For unsexed samples, one to two ejaculate

639 per bull were used (n = 4) for seven IVF replicates. In the case of sexed samples, one to

640 two ejaculate per bull were used (n = 2) in three IVF replicates.

641
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642 Supplementary Table 1. Percentage of induced and spontaneous acrosome

643 reaction (AR) determined for each treatment and type of sperm sample.

644

645
Sperm Treatment Induced AR Spontaneous AR

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646 sample (%) (%)
Before SSA 29±4a 20±4b
647 Unsexed After SSA without P 35±6 a 26±6 b

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After SSA with P 45±6 a 29±5 b
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Before SSA 32±4 a 23 ±4 b
649 Sexed After SSA without P 27±3 a 20±2 b

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After SSA with P 36±1 a 22±1 b
650

651 Data are expressed as mean±SEM. Different letters indicate significant differences

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652 between induced AR vs. spontaneous AR for each treatment as revealed by the t test, p <
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653 0.05.

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