Highlights

Highlights

The natural antioxidants crocin and curcumin were tested as supplements for freezing bull semen
with the commercial extender BIOXCell, with GSH 0.5 mM as reference.
None of the antioxidant formulations clearly benefit sperm quality after thawing or incubation.
Crocin (0.5 and 1.5 mM) exerted mostly a negative effect on sperm physiology.
Curcumin (0.05 and 0.1 mM) showed little effects but stimulated the production of cytoplasmic ROS.
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1 Supplementation of the BIOXcell extender with the antioxidants crocin, curcumin and GSH

1 2 for freezing bull semen

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6 4 Amer Salman1, J. Néstor Caamaño2, Estela Fernández-Alegre1,3, Carlos O. Hidalgo2, Touba
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8 5 Nadri4, Carolina Tamargo2, Carmen Fueyo2, Ángel Fernández2, María J. Merino2, Felipe
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11 6 Martínez-Pastor1,5*
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16 8 INDEGSAL, Universidad de León, 24071 León, Spain
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18 9 SERIDA, 33394 Gijón, Spain
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10 Bianor Biotech SL, 24007 León, Spain
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23 11 Department of Animal Science, College of Agriculture and Natural Resource, University of
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25 12 Tehran, Karaj, Iran
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28 13 Molecular Biology (Cell Biology), Universidad de León, 24071 León, Spain
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35 16 * Corresponding author:
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38 17 Felipe Martínez-Pastor
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40 18 felipe.martinez@unileon.es
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 21 Abstract

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2 22 Semen cryopreservation is routine in cattle, but the results of artificial insemination need
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4 23 improvement. A strategy to these aims is the supplementation of the freezing extender with
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24 novel antioxidants. This study aimed at testing the natural antioxidants curcumin and crocin
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9 25 as supplements to the commercial extender BIOXcell for freezing semen from 8 Holstein
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11 26 bulls. We tested curcumin at 0.05 and 0.1 mM (CU0.05, CU0.1) and crocin at 0.5 and 1.5 mM
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14 27 (CR0.5, CR1.5), with 0.5 mM reduced glutathione (GSH0.5) as reference, and a control
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16 28 (CTL, without supplementation). The samples were evaluated post-thawing and after 5 h at
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19 29 38 °C by CASA for motility and flow cytometry for viability, apoptotic, capacitation,
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21 30 acrosomal status, cytoplasmic and mitochondrial reactive oxygen species (ROS) production
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31 and chromatin status (SCSA). Control and GSH0.5 showed similar results, possibly because
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26 32 of the good protection from BIOXcell. CU0.05 and CU0.1 showed little effects, but increased
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28 33 cytoplasmic ROS production and motility ALH. CR0.5 and CR1.5 decreased viability and
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31 34 increased apoptotic features significantly post-thawing and after the incubation, resulting in
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33 35 lower motility (significant after the incubation), but decreasing SCSA %HDS (loose
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36 36 chromatin). Whereas crocin at these concentrations seems incompatible with BIOXcell,
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38 37 maybe because a prooxidant activity, curcumin use merits further research, considering the
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41 38 elevation of ROS with no significant negative effects.
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39 Keywords: Spermatozoa, cattle, cryopreservation, crocin, curcumin, ROS
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 40 1. Introduction
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2 41 Artificial insemination (AI) with cryopreserved semen is a consolidated technology in
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4 42 modern cattle production, especially dairy (Binyameen et al., 2019). However, despite the
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7 43 development of improved protocols to preserve and apply bull semen (Curry, 2000; Thibier
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9 44 and Wagner, 2002), progress has slowed down over the past few decades (Grötter et al., 2019)
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12 45 The post-thawing quality of cryopreserved semen doses is a significant issue to improve
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46 (Curry, 2000). One of the problems faced during semen cryopreservation is the unbalance of
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17 47 the redox system in spermatozoa. Cell stress results in the generation of high levels of reactive
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19 48 oxygen species (ROS) and other oxidizing molecules, contributing to the lower sperm quality
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22 49 and fertility post-thawing (Chatterjee and Gagnon, 2001). A combination of enzymatic and
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24 50 non-enzymatic antioxidants in the seminal plasma, sperm membranes, and cytoplasm limits
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27 51 ROS accumulation, but these defenses are easily overcome in the stressing conditions of
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29 52 sperm cooling, freezing, and thawing (Agarwal et al., 2014). Moreover, the sperm plasma
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32 53 membrane is rich in polyunsaturated fatty acids (PUFAs), rendering them extremely
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34 54 susceptible to oxygen-induced damage, and hence lipid peroxidation (LPO), which amplifies
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55 the initial effects of oxidative stress (Chatterjee and Gagnon, 2001; Ortega Ferrusola et al.,
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39 56 2009).
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42 57 There is an interest in using natural compounds as antioxidant additives in semen
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44 58 extenders to reduce the harmful effects of ROS on sperm. Thus, we have focused our attention
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47 59 on the antioxidants curcumin and crocin as potential supplements to BIOXcell, a commonly
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49 60 used egg yolk-free commercial extender (Tamargo et al., 2019). Curcumin
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52 61 (diferuloylmethane) is a natural polyphenolic antioxidant (Valgimigli and Pratt, 2012) from
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54 62 the spice turmeric (Curcuma longa). Curcumin has anti-inflammatory and other potentially
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63 therapeutic properties from its antioxidant effect (Menon and Sudheer, 2007), and it could
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 64 alleviate the effects of oxidative stress in spermatozoa (Tvrdá et al., 2016; Zhang et al., 2017).

1 65 Several studies have reported positive effects of curcumin for freezing semen from some
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66 domestic species, such as bull (Bucak et al., 2012), buffalo (Shah et al., 2017), goat (Bucak et
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6 67 al., 2010), or boar (Chanapiwat and Kaeoket, 2015).
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9 68 Crocin is a glucosyl ester of crocetin, a water-soluble carotenoid extracted from saffron
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11 69 (Crocus sativus L.) with a high ability to scavenge the free radicals (Esposito et al., 2019),
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14 70 especially superoxide anion (Galano et al., 2010). This antioxidant has shown promising
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16 71 effects for protecting spermatozoa from different ruminants (Domínguez-Rebolledo et al.,
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19 72 2010; Mata-Campuzano et al., 2015; Sapanidou et al., 2015). One study assessed its
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21 73 efficiency as a supplement for freezing goat semen, showing that 1 mM improved post-
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74 thawing motility and decreased DNA fragmentation and cytoplasmic ROS (Longobardi et al.,
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26 75 2020).
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29 76 To the best of our knowledge, there are no previous studies of the use of curcumin for
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31 77 freezing bull spermatozoa in BIOXcell extender, and no reports on crocin use for this purpose.
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34 78 Therefore, we have tested several concentrations of these antioxidants, 0.05 and 0.1 mM for
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36 79 curcumin (Tvrdá et al., 2016), 0.5 and 1.5 mM for crocin (Longobardi et al., 2020; Sapanidou
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39 80 et al., 2015). As a reference, we added a treatment using GSH. This antioxidant has been
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41 81 widely tested for freezing semen (Foote et al., 2002; Tuncer et al., 2010), and there is a report
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82 combining it with BIOXcell (Sariözkan et al., 2009).
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47 83 2. Materials and methods
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50 84 2.1. Reagents and solutions
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53 85 General reagents were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt,
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55 86 Germany). The freezing extender BIOXcell was obtained from IMV (L'Aigle, France).
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 87 Consumables and solutions for flow cytometry were purchased from Beckman Coulter (Brea,

1 88 CA, USA) and Thermo Fisher (Waltham, MA, USA). Fluorescent probes were purchased
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89 from Thermo Fisher, except Hoechst 33342 and propidium iodide, from Sigma-Aldrich.
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7 90 2.2. Animals and sample collection
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10 91 This experiment used semen samples from eight Holstein bulls (trained animals
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12 92 subjected to a routine collection for semen freezing and AI) allocated at the Artificial
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93 Insemination Center (Centro de Selección y Reproducción Bovina de Cenero, SERIDA).
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17 94 Semen was obtained by artificial vagina (42 °C to 46 °C, non-spermicidal lubricant). All of
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19 95 the animals were in regular AI service and were housed in half-open barns (one bull in each
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22 96 separate barn) under natural environments. They were in good health and met the EU
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24 97 requirements for bulls used in artificial insemination centers. All the procedures were carried
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27 98 out as part of the AI center's routine procedures, the semen samples being donated to the
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29 99 researchers, who had no interaction with the animals.
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33 100 2.3. Experimental design
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35 101 We tested the antioxidants curcumin, crocin, and GSH as supplements to the freezing
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38 102 extender for improving the post-thawing bull semen quality. We split the ejaculates into the
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40 103 control (without supplementation) and supplemented the extender with curcumin 0.05 and
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43 104 0.1 mM, crocin 0.5 and 1.5 mM, and GSH 0.5 mM, and then the semen was frozen. The post-
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45 105 thawing semen was evaluated by CASA and flow cytometry immediately after thawing and
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106 after five incubation hours at 38 °C.
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51 107 2.4. Semen processing
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54 108 After semen collection, the ejaculate volume was estimated directly from the graduated
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56 109 tubes, while the concentration was determined by absorbance with an adapted photometer
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 110 calibrated for bull semen (Accucell, IMV Technologies, L'Aigle, France). Before the

1 111 extension, two concentrations of curcumin (100 µM and 200 µM), two concentrations of
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112 crocin (1 mM and 3 mM), and one concentration of GSH (1 mM) were prepared using
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6 113 BIOXcell at room temperature. Semen pre-diluted with BIOXcell (184×10 6/ml) was split into
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8 114 six aliquots in 15-ml tubes, adding another volume of BIOXcell (Control) or supplemented
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11 115 BIOXcell to the corresponding tubes, achieving a sperm concentration of 92×106/ml.
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13 116 Therefore, we obtained six treatment tubes: Control, 0.05 mM (CU0.05) and 0.1 mM (CU0.1)
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16 117 curcumin, 0.5 mM (CR0.5) and 1.5 mM (CR1.5) crocin, and 0.5 mM (GSH0.5) GSH. These
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18 118 tubes were left to cool at 5 °C for ninety minutes, then equilibrated in a cooler at 5 °C for four
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119 hours. The sperm doses (in 0.25-ml straws) were frozen in liquid nitrogen vapors using a
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23 120 programmable freezer (Digit-cool; IMV Technologies). The freezing followed a standard
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25 121 curve for bovine semen (Muiño et al., 2008): -5 °C/min from +4 °C, to -10 °C; -40 °C/min
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28 122 from -10 °C, to -100 °C, and -20 °C/min from -100 °C, to -140 °C, and then stored in liquid
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30 123 nitrogen. Thawing (two straws, subsequently pooled) was completed in a water bath at 37 °C
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33 124 for thirty seconds.
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36 125 2.5. Determination of sperm motility by CASA (Computer Assisted Sperm Analysis)
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39 126 To analyze the motility and kinematic parameters, we loaded a 10-µm deep Makler®
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41 127 sperm counting chamber (Sefi-Medical Instruments, Haifa, Israel) with 5 µl of sample
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44 128 prediluted in PBS-0.5% BSA at 30×106/ml. It was examined under a microscope (Nikon
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46 129 E400, Tokyo, Japan) with a ×10 negative phase contrast objective and a Basler A312fc camera
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49 130 (Basler Vision Components, Ahrensburg, Germany). At least three fields were captured at
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51 131 53 frames/s, with around two hundred sperm per field. Images were analyzed using the
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132 ISAS® v. 1.019 system (Proiser R+D, Paterna, Valencia, Spain), providing kinetic parameters
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56 133 for each spermatozoon: VCL (curvilinear velocity); VSL (straight path velocity); VAP
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 134 (average path velocity); LIN (linearity, VSL/VCL); STR (straightness, VSL/VAP); WOB

1 135 (wobble, VAP/VCL); ALH (amplitude of the lateral displacement of the sperm head); BCF
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136 (beat-cross frequency of the flagellar beat); DNC (sperm dance, ALH×VCL); DNCm: (sperm
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6 137 mean dance, ALH×VCL/VSL). The data obtained by the CASA system were summarized by
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8 138 extracting the median values for each sample and obtaining the proportion of motile
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11 139 spermatozoa (MOT) and progressive spermatozoa (PROG, STR>80%). A sperm
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13 140 subpopulation analysis was performed by applying a two-step cluster algorithm procedure
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16 141 described previously (Gallego et al., 2012; Ledesma et al., 2017). This analysis allows a
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18 142 grouping of the motility data to classify the sperm in groups characterized by distinctive
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143 motility patterns.
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24 144 2.6. Flow cytometry analysis
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27 145 Flow cytometry analyses of the functional parameters of bull sperm were carried out
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29 146 using a CyAn ADP flow cytometer (Beckman Coulter, Inc., Brea, USA). The sperm samples
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32 147 were incubated in 300 µL of PBS 0.5% BSA with different fluorescent probes (prepared the
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34 148 same day), at 1.7×106/ml, and incubated at 38 °C for 15 min in the dark.
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37 149 The combinations of fluorescent probes to enable the assessment of different
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39 150 physiological variables were: 4.5 µM Hoechst 33342 (H342), 100 nM YO-PRO-1 (YP), 2 µM
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42 151 Merocyanine 540 (M540), 3 µM propidium iodide (PI), and 100 nM MitoTracker deep red
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44 152 (MT) for debris identification, apoptosis, capacitation, viability, and mitochondrial activity,
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47 153 respectively; 4.5 µM Hoechst 33342 (H342), 1 µg/mL peanut agglutinin conjugated with
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49 154 FITC (PNA), and 3 µM propidium iodide (PI) for debris identification, viability and
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52 155 acrosomal status; and 4.5 µM Hoechst 33258 (H258), 5 µM CM-H2DCFDA (CFDA), and
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54 156 1 µM MitoSOX (MSX) for viability, cytoplasmic ROS, and mitochondrial superoxide
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157 production. The configuration of the cytometer was: Violet laser line (405 nm) with a 450/50
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 158 filter for H342; blue laser line (488 nm) with a 530/40 filter for YO-PRO-1, PNA, and CFDA,

1 159 a 575/25 filter for M540 and a 613/20 filter for PI and MSX; red laser line (633 nm) with a
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160 665/20 filter for MT and PNA. The acquisition was controlled with the Summit software v.
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6 161 4.3.02 (Beckman Coulter), acquiring at least 5000 events identified as spermatozoa (FSC/SSC
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8 162 and H342 when available for sperm gating). Data were saved as FCS v.3 files and analyzed
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11 163 with the Weasel v. 3.5 software (Frank Battye, Melbourne, Australia). We obtained the
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13 164 following variables as %: Total viability as PI – events, with viability/non-apoptotic as YP–/PI–;
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16 165 total acrosomal damage as PNA+, with acrosomal damage ratio as PNA+ within the PI–
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18 166 population; apoptosis ratio as YP+ within the PI- population; capacitation ratio as M540+ in the
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167 YP– population; mitochondrial activity as MT+/YP– spermatozoa; mitochondrial superoxide
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23 168 production ratio as MSX+ within the H258– population. The mean fluorescence intensity
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25 169 (MFI) of CFDA (within the H258– population) was used for estimating cytoplasmic ROS
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28 170 production.
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31 171 2.7. Assessment of sperm chromatin integrity
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34 172 We used the SCSA (Sperm Chromatin Structure Assay) to assess sperm DNA integrity
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173 and chromatin compaction. We diluted the sperm sample (directly post-thawing and after
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39 174 incubation) in TNE buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 7.4) to 2×106/
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41 175 ml, and stored the samples at -80 °C. The samples were thawed on crushed ice before
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44 176 analysis. A volume of 200 µl was pipetted in a flow cytometry tube and immediately mixed
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46 177 with 400 µl of acid-detergent solution (0.1% Triton X-100, 150 mM NaCl, and 80 mM HCl,
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49 178 pH 1.2) to induce denaturation of DNA in situ. Exactly thirty seconds later, the sample was
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51 179 stained by adding 1.2 ml of acridine orange (AO) solution (0.1 M citric acid, 0.2 M Na2HPO4,
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180 1 mM disodium EDTA, and 0.15 M NaCl, pH 6.0; 6 μg/ml AO). The tube was kept in crushed
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56 181 ice and run in the cytometer after three minutes. We used a FACSCalibur flow cytometer
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 182 (Becton Dickinson; Franklin Lakes, NJ) with a blue laser (488 nm) and filters for green

1 183 (530/30) and red (650LP) fluorescence of the AO. At least 5000 spermatozoa were analyzed
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184 in each run. AO emits red fluorescence if it binds to single-strand DNA (susceptible to
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6 185 denaturation, with breaks) and green fluorescence if it binds to double-strand DNA (resistant
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8 186 to denaturation, no breaks). The DNA fragmentation index (DFI) was calculated for each
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11 187 spermatozoon as the ratio of red to total (red+green) fluorescence, times 1000. We obtained
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13 188 the standard deviation of the DFI (SD-DFI) and the spermatozoa proportion with a DFI higher
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16 189 than 250 (%DFI). Chromatin compaction was estimated from the green fluorescence intensity,
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18 190 considering the spermatozoa with higher intensity than 650 (%HDS).
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22 191 2.8. Statistical analysis
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24 192 Statistical analyses were carried out in the R statistical environment (R Team, 2019).
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27 193 The data was analyzed using linear-mixed effects models. The antioxidant treatment and the
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29 194 incubation time, and their interaction, were included in the fixed part of the model, while the
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32 195 bull was the grouping factor in the random part.
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35 196 3. Results
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38 197 The antioxidants curcumin and crocin for bull semen cryopreservation, with glutathione
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40 198 as a reference, yielded mixed results. These treatments did not significantly affect the CASA
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43 199 variables post-thawing, either overall motility (Fig. 1) or the kinematic parameters (Fig. 2).
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45 200 Only after the incubation, MOT, PROG (Fig. 1), and LIN (Fig. 2d) significantly declined with
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201 both crocin concentrations. LIN also decreased with GSH, and WOB (Fig. 2f) decreased in all
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50 202 the treatments. Crocin even abated BCF (Fig. 2h) after the incubation and increased DNCm
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52 203 (Fig. 2i; reflecting linearity loss). Curcumin 0.05 mM slightly increased ALH after the
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55 204 incubation (Fig. 2g), and this treatment and GSH 0.5 mM also increased DNCm (Fig. 2i).
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 205 However, we did not notice changes in the motility subpopulations obtained after the

1 206 clustering analysis. The two-step procedure yielded two subpopulations, Rapid and Slow
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207 (Table 1). We found a noticeable variability between males, and neither the treatments nor the
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6 208 incubation significantly affected the proportions of these subpopulations (Fig. 3).
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9 209 Flow cytometry analysis, being more sensitive, showed many changes in sperm
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11 210 physiological parameters just after thawing (Fig. 4). Crocin caused an evident significant
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14 211 depression in sperm viability measured by propidium iodide (loss of membrane integrity;
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16 212 Fig. 4a) or YO-PRO-1 (membrane integrity and apoptotic-like features; Fig. 4b) post-thawing,
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19 213 at both concentrations and with similar effects. Both crocin concentrations also induced
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21 214 several physiological changes post-thawing: A surge in the proportion of viable spermatozoa
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215 with apoptotic features (YO-PRO-1+/PI–; Fig. 4c) and the apoptotic ratio (YO-PRO-1+
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26 216 spermatozoa within the PI– subpopulation; Fig 4d); and decreased the proportion of
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28 217 spermatozoa with active mitochondria, both overall (Fig. 4g) and as a ratio of the viable
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31 218 (YO-PRO-1–) population (Fig. 4j). These effects were also present after the incubation, except
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33 219 for the apoptotic's population change, possibly due to the overall loss of viability. Crocin did
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36 220 not affect acrosomal integrity after thawing, but after the incubation, it caused a significant
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38 221 increase in the proportion of acrosome-damaged spermatozoa (overall PNA +; Fig. 4f).
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41 222 Contrarily, curcumin, and GSH did not significantly affect these physiological variables. Only
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43 223 both curcumin concentrations slightly increased (P<0.01) the acrosomal damage considering
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45 224 the ratio in the viable subpopulation PI– (Fig. 4g), only in the post-thawing assessment.
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48 225 Interestingly, the antioxidant treatments showed a very different effect on sperm ROS
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51 226 production (Fig. 5). Curcumin considerably increased cytoplasmic ROS presence both post-
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53 227 thawing and after the incubation (Fig. 5a), whereas these levels remained basal in the other
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56 228 treatments. Whereas the proportion of spermatozoa with high mitochondrial superoxide
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 229 remained very low in all cases (Figures 5b and 5c), curcumin 0.1 mM had a decreasing effect

1 230 (significant when considering the H258–/MSX+ subpopulation, but not as ratio within the
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231 H258– population). GSH 0.5 mM significantly decreased both variables, whereas crocin
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6 232 1.5 mM significantly increased the mitochondrial superoxide production, but only when
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8 233 assessing post-thawing.
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11 234 In the assessment of chromatin alterations by SCSA (Fig. 6), we found an excellent
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14 235 condition both in the thawed and incubated samples, with only a small proportion of
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16 236 spermatozoa with DNA fragmentation or chromatin immaturity (0.8% ± 0.5 and 4.1% ± 1.7
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19 237 respectively; mean ± SD). Only crocin decreased %HDS (P<0.001 for 1.5 mM at both times
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21 238 and P<0.01 for 0.5 mM after the incubation; Fig. 6c), with crocin 0.5 mM slightly reducing
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239 DFI (DNA fragmentation index) heterogeneity after the incubation (P<0.05; Fig. 6a).
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27 240 4. Discussion
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30 241 Curcumin and crocin are natural antioxidants with excellent ROS scavenging properties
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32 242 and could preserve the endogenous antioxidants in semen samples (Domínguez-Rebolledo et
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243 al., 2010; Tvrdá et al., 2016). Nevertheless, we did not find evidence of a positive effect on
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37 244 the post-thawing quality of bull spermatozoa. However, some outcomes could be useful when
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39 245 applying antioxidant supplements to commercial extenders such as BIOXcell.
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42 246 Previous studies applied these antioxidants to self-made extenders devoid of
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45 247 antioxidants (except some activity provided by components such as Tris or those contained in
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47 248 egg yolk). Some authors reported improving post-thawing sperm quality by using curcumin to
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50 249 supplement Tris-citric-fructose-egg yolk (TCFY) extenders. Thus, curcumin decreased sperm
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52 250 abnormalities and enhanced sperm quality post-thawing in bull (Bucak et al., 2012) and
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55 251 buffalo (Shah et al., 2017).
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 252 One reason for these differences could be the interaction of the antioxidants with the

1 253 extender. Our study aimed to test the supplements with the BIOXcell extender since this is
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254 one of the most widely used for freezing bull semen. Most breeding centers use commercial
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6 255 extenders because they enable standardization and homogeneity among batches and sanitary
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8 256 and quality assurance reasons. However, the composition of these extenders is proprietary,
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11 257 and therefore differences with other studies using self-made extenders are expected. Indeed,
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13 258 Stradaioli et al. (2007) found that BIOXcell was very efficient in maintaining GSH levels in
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16 259 the cryopreserved semen samples due to its antioxidant content. These authors reported an
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18 260 equivalent content of 450 µM GSH in this extender, which could explain why the GSH
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261 treatment used as a reference in this study showed little effects (and the low levels of
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23 262 cytoplasmic and mitochondrial ROS in our control samples). In this regard, there is some
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25 263 controversy on the effectiveness of GSH supplementation for improving the performance of
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28 264 frozen bull semen (Foote et al., 2002; Shah et al., 2017).
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31 265 Additionally, our study used curcumin doses in the low micromolar, as reported by
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33 266 Tvrdá et al. (2016). However, Bucak et al. (2012), using TCFY, showed an improvement in
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36 267 the post-thawing bull semen quality using 0.5 mM, while they did not find such increases with
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38 268 2.0 mM. These observations are compatible with a good performance of BIOXcell in our
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41 269 study, which might have masked any improvement due to additional antioxidants. Whereas
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43 270 future studies could confirmed these hypotheses, a higher antioxidant concentration could not
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45 271 be beneficial. Bucak et al. (2012) noticed a lower performance with higher concentrations, but
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48 272 studies on other species have reported adverse effects with low curcumin doses. Thus, Naz
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50 273 (2011) showed that curcumin at only 125 µM led to a sharp decrease in forward sperm
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53 274 motility in human and murine spermatozoa, with detrimental effects even at lower
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55 275 concentrations.
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 276 Interestingly, curcumin induced an increase in cytoplasmic ROS post-thawing and after

1 277 incubation, both at 10 and 50 µM. Some authors reported that curcumin, despite to its
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278 antioxidant properties, may also increase the generation of ROS by a direct action or by
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6 279 reducing the endogenous GSH (Atsumi et al., 2007; Banerjee et al., 2008; Chen et al., 2005;
7
8 280 Sánchez et al., 2010). Indeed, curcumin has been proposed as a cytotoxic therapy for cancer
9
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11 281 cells by causing an increase in the generation of ROS (Chen et al., 2005; Sánchez et al.,
12
13 282 2010). Atsumy et al. (2007) reported that the incubation of the cells for one hour with
14
15
16 283 curcumin at 10 µM resulted in the same levels of GSH as in the control (without curcumin
17
18 284 addition), but it induced an important drop in these levels when curcumin was present at
19
20
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285 30 µM. These effects could be related to the spermicidal activity reported by Naz (2011).
22
23 286 However, Bucak et al. (2012) pointed out that curcumin at 0.5 mM allowed the maintenance
24
25 287 of glutathione levels in post-thawed bull sperm.
26
27
28 288 In our study, this increase of cytoplasmic ROS was not accompanied by any negative
29
30
31 289 effects on sperm quality comparing to the control, not even after the 5 h incubation. This is
32
33 290 interesting and merits further research. Whereas ROS have been associated with negative
34
35
36 291 effects, they are important as transducers of intracellular signals (Aitken, 2011; Baskaran et
37
38 292 al., 2020). Therefore, a sustained increase in intracellular ROS could benefit sperm
39
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41 293 performance, especially if an external source of reducing power supports the intracellular
42
43 294 antioxidant system (e.g. GSH or similar in BIOXcell). Moreover, the curcumin at 0.1 mM
44
45 295 also slightly reduced the proportion of viable spermatozoa with high mitochondrial
46
47
48 296 superoxide production. A dual activity of curcumin in the spermatozoa could explain these
49
50 297 contradictory reports, with curcumin activity depending not only on concentration or cell type
51
52
53 298 but also on the environment composition.
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 299 Contrarily, crocin led to a decline in sperm quality, more noticeably in flow cytometry

1 300 parameters after thawing, reflecting in a motility decline after incubation. Crocin is an
2
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301 effective antioxidant, with a high ability to scavenge free radicals (Esposito et al., 2019;
5
6 302 Galano et al., 2010; Sapanidou et al., 2016). Whereas other authors have obtained good results
7
8 303 by using crocin as a post-thawing supplement for bull sperm (Sapanidou et al., 2015), our
9
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11 304 study is the first reporting the use of this antioxidant as a supplement for freezing. These
12
13 305 differences are not due to different concentrations or species (although Sapanidou et al.
14
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16 306 reported using a commercial extender, whose name was not disclosed), bur rather to
17
18 307 alternative activities of crocin depending on the funcional status of the spermatozoa or to the
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308 activity of stressors such as the freezing/thawing processes.
22
23
24
309 We reported a similar situation with other antioxidants, such as the well-known Trolox.
25
26 310 Trolox yielded very good results by protecting thawed red deer spermatozoa from oxidative
27
28 311 stress (Domínguez-Rebolledo et al., 2010), but depressed motility when used as a supplement
29
30
31 312 for the freezing extender while maintaining excellent antioxidant properties (Anel-López et
32
33 313 al., 2012). Thus, one reason for the detrimental effects of crocin in our experiment could be an
34
35
36 314 excessive antioxidant effect in key instances of the freezing/thawing process, maybe
37
38 315 impairing signaling pathways related to sperm viability (de Lamirande and O’Flaherty, 2008;
39
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41 316 Sanocka and Kurpisz, 2004). This could be the same when testing other potent antioxidants
42
43 317 (Mata-Campuzano et al., 2012a, 2012b). However, there is another hypothesis more apt for
44
45 318 the case of crocin. Just as curcumin, crocin seems to have a dual activity as anti/prooxidant,
46
47
48 319 possibly occurring simultaneously. In our study with thawed red deer spermatozoa
49
50 320 (Domínguez-Rebolledo et al., 2010), crocin was very efficient stimulating sperm motility,
51
52
53 321 even in a situation with induced oxidative stress. However, we observed a dual action of
54
55 322 crocin, decreasing intracellular ROS and DNA damage in presence of exogenous oxidative
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 323 stress but increasing intracellular ROS and lipid peroxidation during a normal incubation.

1 324 Interestingly, crocin reduced %HDS in a manner compatible with increased oxidative activity.
2
3
4 325 In another experiment, freezing ram semen with 1 mM crocin did not result in lower
5
6
326 motility and protected sperm chromatin, although we detected an increase in acrosomal
7
8
9 327 damage (Mata-Campuzano et al., 2015). Crocin at the same concentration could be effective
10
11 328 for freezing goat semen (Longobardi et al., 2020). This antioxidant could have a different
12
13
14 329 effect in these small ruminants, or show other activity depending on the extender (TCFY or
15
16 330 similar in these studies).
17
18
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20 331 5. Conclusions
21
22 332 The supplementation of curcumin to the BIOXcell extender does not result in a clear
23
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25 333 advantage, at least in the concentrations tested in the present study. Some effects such as the
26
27 334 increase in cytoplasmic ROS deserve further investigation. Crocin showed negative effects on
28
29
30 335 the thawed and incubated samples, and could not be compatible with BIOXcell or freezing
31
32 336 bull semen at the millimolar range.
33
34
35 337 Ethical standards
36
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338 This article does not contain any studies with animals performed by any of the authors.
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339 Declaration of Competing Interest
41
42
43 340 The authors declare that there is no conflict of interest.
44
45
46 341 Acknowledgments
47
48 342 This study was partly supported by grant RZP2017-00008-00-00 (INIA, MINECO,
49
50
51 343 Spain) and was carried out in a collaboration promoted by grant AGL2016-81890-REDT
52
53 344 (MINECO, Spain). We thank SERIDA for providing the semen samples used in this study,
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 345 and Laura Álvarez Fernández, Ender Yalcinkaya Kalyan, Beatriz de Arriba Ruiz, and Lucía

1 346 Tejerina García.

2
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Figure

(a) Total motility (MOT) (b) Progressive motility (PROG)

Figure 1. Effect of the antioxidant treatments (CTL: Control; CR0.5, CR1.5: Crocin 0.5 and 1.5 mM;
CU0.05, CU0.1: Curcumin 0.05 and 0.1 mM; GSH0.5: GSH 0.5 mM) on motility variables. The plots
show mean ± SEM, directly post-thawing (0 h), and after the 5-h incubation (5 h). Insets show P
values for the effects of the treatments respect to the Control (* P<0.05; ** P<0.01). The overall
effect of the incubation was P<0.05 for MOT.
(a) VCL (b) VAP (c) VSL

(d) LIN (e) STR (f) WOB

(g) ALH (h) BCF (i) DNCm

Figure 2. Effect of the antioxidant treatments (CTL: Control; CR0.5, CR1.5: Crocin 0.5 and 1.5 mM;
CU0.05, CU0.1: Curcumin 0.05 and 0.1 mM; GSH0.5: GSH 0.5 mM) on CASA kinematic variables.
The plots show mean ± SEM for kinematic variables (continued to Fig. 2b), directly post-thawing
(0 h), and after the 5-h incubation (5 h). Insets show P values for the effects of the treatments respect
to the Control (* P<0.05; ** P<0.01; *** P<0.001). The overall effect of the incubation was not
significant except for VCL and WOB (except the control).
 (j) Rapid (k) Slow

Figure 3. Effect of the antioxidant treatments (CTL: Control; CR0.5, CR1.5: Crocin 0.5 and 1.5 mM;
CU0.05, CU0.1: Curcumin 0.05 and 0.1 mM; GSH0.5: GSH 0.5 mM) on CASA variables. The plots
show mean ± SEM for sperm subpopulations, directly post-thawing (0 h), and after the 5-h incubation
(5 h). Neither the treatment nor the incubation time significantly affected the variables.
(a) Viability (PI–) (b) Viability (YO-PRO-1–) (c) Apoptotic (YO-PRO-1+/PI–)

(d) Apoptotic (ratio viable) (e) Capacitated (M540+, ratio viable) (f) Acrosomal damage (PNA+, total)

(g) Acrosomal damage (ratio viable) (h) Mitochondrial activity (MT+) (i) Mit. activity (ratio viable)

Figure 4. Effect of the antioxidant treatments (CTL: Control; CR0.5, CR1.5: Crocin 0.5 and 1.5 mM;
CU0.05, CU0.1: Curcumin 0.05 and 0.1 mM; GSH0.5: GSH 0.5 mM) on sperm physiology as
assessed by flow cytometry. The plots show mean ± SEM, directly post-thawing (0 h), and after the
5-h incubation (5 h). Variables showing ratio viables are transformed as the ratio of positive events
within the viable population (YO-PRO-1– or PI– depending on the case). Insets show P values for the
effects of the treatments respect to the Control (* P<0.05; ** P<0.01; *** P<0.001). The overall
effect of the incubation was P<0.001 except for capacitation ratio. M540: Merocyanine 540; PNA:
Peanut agglutinin, FITC conjugated; MT: Mitotracker deep red.
(l) Cytoplasmic ROS (CFDA+, viable) (m) Mitochondrial O2•- (H258–(n) Mitochondrial O2•- (ratio viable)
/MSX+)

Figure 5. Effect of the antioxidant treatments (CTL: Control; CR0.5, CR1.5: Crocin 0.5 and 1.5 mM;
CU0.05, CU0.1: Curcumin 0.05 and 0.1 mM; GSH0.5: GSH 0.5 mM) on ROS production variables,
assessed by flow cytometry. The plots show mean ± SEM (continued from Fig. 3a), directly post-
thawing (0 h), and after the 5-h incubation (5 h). Insets show P values for the effects of the treatments
respect to the Control (* P<0.05; ** P<0.01; *** P<0.001). The overall effect of the incubation was
P<0.001, except P<0.01 for cytoplasmic ROS. CFDA: CM-H2DCFDA H258: Hoechst 33258; MSX:
MitoSOX.
(o) SD-DFI (p) DNA fragmentation (%DFI) (q) Chromatin immaturity (%HDS)

Figure 6. Effect of the antioxidant treatments (CTL: Control; CR0.5, CR1.5: Crocin 0.5 and 1.5 mM;
CU0.05, CU0.1: Curcumin 0.05 and 0.1 mM; GSH0.5: GSH 0.5 mM) on the chromatin structure
variables obtained by SCSA. The plots show mean ± SEM, directly post-thawing (0 h), and after the
5-h incubation (5 h). Insets show P values for the effects of the treatments respect to the Control (*
P<0.05; ** P<0.01; *** P<0.001). The overall effect of the incubation was P<0.001 except for %DFI
with P>0.05. DFI: DNA fragmentation index.