Antioxidant effect of crocin on bovine sperm quality and in vitro fertilization

Accepted Manuscript Antioxidant effect of crocin on bovine sperm quality and in vitro fertilization V. Sapanidou, I. Taitzoglou, Ι. Tsakmakidis, I. Kourtzelis, D. Fletouris, A. Theodoridis, I. Zervos, M. Tsantarliotou PII:

S0093-691X(15)00347-7

DOI:

10.1016/j.theriogenology.2015.07.005

Reference:

THE 13251

To appear in:

Theriogenology

Received Date: 4 March 2015 Revised Date:

1 July 2015

Accepted Date: 1 July 2015

Please cite this article as: Sapanidou V, Taitzoglou I, Tsakmakidis Ι, Kourtzelis I, Fletouris D, Theodoridis A, Zervos I, Tsantarliotou M, Antioxidant effect of crocin on bovine sperm quality and in vitro fertilization, Theriogenology (2015), doi: 10.1016/j.theriogenology.2015.07.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Revised non-highlighted

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Antioxidant effect of crocin on bovine sperm quality and in vitro

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fertilization

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Sapanidou V.1, Taitzoglou I.1, Tsakmakidis Ι.2, Kourtzelis I.3, Fletouris D.4,

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Theodoridis A.5, Zervos I.1, Tsantarliotou M.1, *

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Sciences, 54124, Aristotle University of Thessaloniki, Greece

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Laboratory of Physiology, School of Veterinary Medicine, Faculty of Health

Clinic of Farm Animals, School of Veterinary Medicine, Faculty of Health

Sciences, 54124, Aristotle University of Thessaloniki, Thessaloniki, Greece

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Aristotle University of Thessaloniki, Greece

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Veterinary Medicine, Faculty of Health Sciences, 54124, Aristotle University

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of Thessaloniki, Greece

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Faculty of Health Sciences, 54124, Aristotle University of Thessaloniki,

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Thessaloniki, Greece

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Laboratory of Genetics and Molecular Biology, School of Biology, 54124,

Laboratory of Hygiene and Technology of Food Animal Origin, School of

Laboratory of Animal Production Economics, School of Veterinary Medicine,

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Medicine, Faculty of Health Science, 54124, Aristotle University of

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Thessaloniki, Greece, [email protected]

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Keywords: crocin, sperm, embryos, antioxidants, oxidative stress

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Corresponding author: Laboratory of Physiology, School of Veterinary

Abstract

Reactive Oxygen Species (ROS) production above critical levels affects the

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genetic and functional integrity of spermatozoa by causing oxidative stress.

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Spermatozoa are susceptible to oxidative stress in terms of motility and fertilization

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capacity. Crocin (crocetin di-gentiobiose ester), a main constituent of Crocus Sativus

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L. (saffron) is known for its antioxidant activity, by scavenging ROS, especially

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superoxide anion. The aim of the present study is to evaluate the effect of crocin on

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the quality characteristics of spermatozoa and fertilization rate. Frozen/thawed and

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ACCEPTED MANUSCRIPT washed spermatozoa from 4 different bulls were incubated with 3 different

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concentrations of crocin (0.5mM, 1mM and 2mM), for 120 min and 240 min

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respectively, in the presence of a negative control, and was evaluated in terms of

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motility, viability, acrosomal status, DNA fragmentation index, intracellular ROS and

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lipid peroxidation. The most potent concentration of crocin (1mM) was also added in

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the fertilization medium to test its impact on fertilization outcome. The results indicate

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that the incubation of spermatozoa with 1mM of crocin resulted in a statistically

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significant lower production of ROS, lower lipid peroxidation and in better

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maintenance of motility, viability and acrosomal integrity, with a very small number of

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fragmented cells, compared to the control and the other treated groups (P<0.05).

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1mM of crocin resulted in a significant increase of blastocyst rate, compared to the

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control group (P<0.01). These data indicate that crocin (1mM) improves bovine

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sperm quality and its fertilization capability, directly and/or indirectly, by modulating

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ROS concentration.

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1. Introduction

One of the most important factors contributing to poor semen quality is oxidative

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stress (OS). OS is a condition associated with an imbalance between the production

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of Reactive Oxygen Species (ROS) and the ability of a biological system to detoxify

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the reactive intermediates or easily repair the resulting damage [1]. In semen,

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potential sources of ROS are dead and abnormal/immature spermatozoa, as well as

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the leukocytes that are present in the ejaculate [2,3]. During in vitro fertilization, the

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PO2 is much higher than the PO2 in vivo [4]. Spermatozoa generate superoxide anion

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and hydrogen peroxide, which is formed either spontaneously or through the action

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of superoxide dismutase. Gametes and embryos are very vulnerable to OS,

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especially under in vitro conditions. It is suggested that mild and low OS may

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enhance the fertilizing potential by promoting hyperactivation, motility and

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capacitation, through increased tyrosine phosphorylation [5,6]. Apart from that, ROS

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mediate crucial reproductive processes, such as sperm-oocyte interaction,

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implantation and early embryo development [6]. An excess production of ROS is

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detected after cryopreservation and thawing or centrifugation; this affects not only

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sperm motility and its ability to fuse the oocyte, but DNA integrity and fertilizing

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capacity, as well [7]. Spermatozoa are particularly susceptible to oxidative injury due

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to the abundance of plasma membrane PUFAs (polyunsaturated fatty acids) [8].

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These unsaturated fatty acids provide fluidity that is necessary for sperm motility [6]

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and for membrane fusion events (e.g., acrosome reaction-AR and sperm–egg

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interaction). However, the free radical attack and the ongoing lipid peroxidation (LPO)

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ACCEPTED MANUSCRIPT throughout the sperm plasma membrane result in accumulation of lipid peroxides on

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the sperm surface, loss of sperm motility [9] and oxidative damage to DNA [10].

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There is evidence that a positive correlation between DNA damage, ROS generation

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and apoptosis exists [7]. An early apoptotic feature reported in spermatozoa is the

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externalization of phosphatidylserine (PS) on the outer leaflet of plasma membrane

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[11], which is associated with a decreased ability to fertilize [12], although Martin and

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co-authors [13] supported that PS exposure in human sperm is mainly related to the

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AR rather than apoptosis.

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Spermatozoa are not heavily equipped with antioxidant systems, capable of

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protecting them from the overwhelming production of ROS. These limitations are due

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to the small volume of cytoplasm, as well as the low concentration of scavenging

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enzymes [14]. Furthermore, the antioxidant systems of the seminal plasma are

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removed during assisted reproductive techniques (ART) [3].

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Spermatozoa undergo the risk of OS and many antioxidants have been employed in

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vitro in order to maintain their integrity and functionality [15,16]. Several experimental

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and clinical studies on pathophysiology of OS and its impact on infertility have

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demonstrated the beneficial role of many antioxidants on sperm parameters and

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pregnancy rates (for a review, see 15,17). Enzymatic antioxidants, such as

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superoxide dismutase, and vitamins (e.g. vitamin E) are regarded as very efficient

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antioxidant agents in order to maintain a stable ratio between OS and the antioxidant

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capacity of spermatozoa [18]. A special group of antioxidants consists of plant-

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derived compounds, such as carotenoids. There is strong evidence that natural

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antioxidants, carotenoids included, may reduce or prevent many diseases that are

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ROS-mediated (e.g. cancer, diabetes, varicocele) [19].

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Carotenoids are equipped with an extensive system of conjugated double edge

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bonds. They are regarded as one of the most efficient 1O2 quenchers, as well as

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ROS scavengers operating in cellular lipid bilayers. Moreover, carotenoids offer a

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special protection against LPO [20].

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Saffron (Crocus sativus, L.) is a natural food additive with a well known antioxidant

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action [21] and multiple therapeutic properties that have been proved both in vitro

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and in vivo [22,23]. Saffron affects positively sperm morphology and motility in

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infertile men [24] and in mice [25].

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Crocin (crocetin di-gentiobiose ester), a main constituent of saffron, is one of the

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few water soluble carotenoids found in nature, which acts as an antioxidant by

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quenching free radicals, especially superoxide anion [26]. Under in vitro conditions

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crocin had an ameliorative effect on post-thawed sperm motility of red deer through

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an optimum level of ROS [27]. Crocin, might affect sperm physiology through its

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protective antioxidant effect in the media of ART. Although there is strong evidence that the use of antioxidant additives enhances

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sperm quality parameters [15,16,17,27], the effect of antioxidant supplementation in

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the IVF medium on fertilization rate and embryo quality remains controversial [28, 29]

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Therefore, the present study was undertaken to investigate for the first time whether

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supplementation of in vitro sperm preparation media with crocin prolong thawed

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sperm quality characteristics over time, by preventing them from the oxidative attack

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in freeze/thawing procedures. Furthermore, crocin was tested as a beneficial

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antioxidant in the IVF medium on fertilization process in terms of embryo

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development rate.

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2. Materials and Methods

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2.1 Experimental design

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The semen used in the experiments originated from four different mature

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Simmental bulls of proven fertility, housed at the Center of Artificial Insemination of

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Thessaloniki (National Agricultural Research Foundation, Nea Ionia, Thessaloniki,

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Greece). The semen was collected with an artificial vagina at the same period of the

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year. The collected semen had >70 % initial motility, >75% viability and a total

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concentration of at least 4 x 109 spermatozoa/ ml. The collection and freezing of

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semen were performed under commercial conditions. Semen was diluted with a

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commercial extender (20% Tris-egg yolk, 7% glycerol, 78mM citric acid, 69 mM

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fructose, 50 µg tylosin, 250 µg gentamycin, 150 µg lincomycin, 300 µg spectinomycin

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in each ml of extended frozen semen) and packed into 0.5 ml plastic straws, each

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one containing approximately 50 x 106 spermatozoa/ml. The frozen straws were

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stored in liquid nitrogen (-196ο C). The experiments were conducted under the

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principles of Good Laboratory Practice (GLP). At the beginning of each experiment, 1

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straw from each bull was thawed by immersion in distilled water (37ο C, 40s). The

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straws were immediately pooled into a sterile plastic tube and were subsequently

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washed with the appropriate media for each assessment. All reagents were

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purchased from Sigma Aldrich Co. (Germany), unless otherwise specified.

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Crocin (crocetin digentiobiose ester-17304) of high purity (>99%) was stored as a

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powder at +4ο C in the dark. The stock solution of crocin (10mM) was prepared in

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water for embryo transfer (W1503), split in aliquots and stored at -20o C in the dark.

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In each experiment a fresh diluted solution of crocin was prepared. Before crocin’s

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supplementation an equal volume of medium was removed.

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2.2. Semen evaluation 2.2.1. Motility and Viability assessment The sample was washed with 3x volume of Sperm Talp (100mM NaCl, 3.1mM

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KCl, 25mM NaHCO3, 0.29mM NaH2PO4, 21.6mM Na Lactate, 2mM CaCl2, 1.5mM

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MgCl2, 10mM Hepes supplemented with 0.6 % bovine serum, 1mM sodium pyruvate

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and 50 µg/ml gentamycin in water for embryo tranfer) and centrifuged at 300x g for

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10 min (RT). The procedure was repeated twice. Sperm concentration was

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determined with a haemocytometer (Optik Labor, Grale HDS, New South Wales,

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Australia). The washed pool of spermatozoa was divided in four tubes. One tube

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served as a control (reference value), while the others were supplemented with three

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different concentrations of crocin (0.5mM, 1mM and 2mM) and Sperm Talp was

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added up to a final volume of 100 µl in order to achieve a concentration of

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20x106cells/ml in each tube. This dilution resulted in a good number of spermatozoa

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per field without aggregations [30]. Five µl aliquot of sperm suspension were placed

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in pre-warmed slide and analyzed by Computer Assisted Sperm Analyzer, using

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Integrated Semen Analysis System Software (ISAS MvCo, Valencia, Spain) at three

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different time points (0 min, 120 min, 240 min), showing 9 different parameters

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(Rapid, Medium, Slow, Static, Progressive Motility, Curvilinear Velocity (VCL),

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Straight Line Velocity (VSL), Average Path Velocity, (VAP), Amplitude Lateral Head,

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(ALH). The CASA system consisted of a triocular optical phase microscope (Nicon

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Eclipse C1, Nikon, Tokyo, Japan), equipped with a warming plate (Tokai, Tokyo,

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Japan) at 37ο C and a Baler Scout CCD digital camera (Basler Vision Technologies,

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Ahrensburg, Germany). The camera was connected to a computer. The default

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settings include the following: image capture by 60 frames/second, total of 25 frames

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captured; cell detection with minimum contrast of 80 and medium cell size of 5 pixels;

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a cutoff value for progressive cells of 50 µm/sec for VAP and 70% for medium

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threshold straightness; slow cells recorded as static with a VAP cutoff 25 µm/sec and

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a VSL cutoff 10 µm/sec. In parallel, smears of spermatozoa corresponding to the

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three time points and the three concentrations were stained with eosin Y-nigrosin,

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according to Björndahl et al [31]. Two hundred spermatozoa per slide were examined

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microscopically (x100), in order to evaluate the viability and the acrosomal status.

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The experiment was conducted 8 times.

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2.2.2. Assessment of DNA integrity

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The pooling was prepared as described above while the final concentration of

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spermatozoa was 30x106 cells/ml. The spermatozoa were treated with 3 different

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concentrations of crocin (0.5mM, 1mM and 2mM) in the presence of a negative 5

ACCEPTED MANUSCRIPT control. The integrity of DNA was assessed at three different time points (0 min, 120

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min, 240 min) by the Acridine Orange Test (AOT). Smears of spermatozoa were

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fixed for 4 hours with freshly prepared Carnoy’s solution (3 methanol: 1 crystalloid

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acetic acid) [32]. After fixation, smears of spermatozoa were stained with a solution

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of acridine orange (A6014) and evaluated under epifluorescence microscope

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(490/530nm excitation/barrier filter, Nikon Eclipse C1 Confocal, Tokyo, Japan). Two

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hundred spermatozoa per slide were assessed in ten different optical areas for

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determination of percentage of spermatozoa with denaturated DNA. Sperm with

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normal DNA content present a green fluorescence, whereas sperm with fragmented

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DNA emit fluorescence in a spectrum varying from yellow to red. The experiment was

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conducted 8 times.

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2.2.3. Determination of intracellular ROS levels and PS externalization The pooling was centrifuged with two gradients (45% and 80%) of Percoll (P4937)

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in order to remove the cryoprotectants. After centrifugation (380x g, 25 min, RT), the

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supernatant was carefully removed and the pellet was reconstituted with 2ml of

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Sperm Talp in order to be centrifuged twice (140x g, 10 min, RT). Subsequently

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spermatozoa were incubated with 3 different concentrations of crocin (0.5mM, 1mM,

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2mM), in the presence of a negative control.

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The determination of H2O2, O2 and PS externalization was accomplished by a flow

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cytometry analyzer (Cyflow ML, Partec, Canterbury, UK), equipped with an air-cooled

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Argon ion laser emitting at 488nm, using the FloMax Software (Partec GmbH,

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Münster, Germany). In each measurement, from each sample of 5 x 105 cells, 1 x 104

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spermatozoa were analyzed. Spermatozoa obtained in the plots were gated using a

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forward-angle (FSC) and a side-angle light scatter (SSC) dot plot to gate out debris

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and aggregates.

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DCFH-DA (2’-7’ Dichloro-dihydro-fluorescein diacetate- D6883) and DHE

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(Dihydroethidium- D7008) are specific probes for H2O2 and O2-., respectively. In order

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to prepare the stock solutions DCFH-DA (250 µM) was diluted in methanol, while

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DHE (500 µM) was diluted in DMSO. The solvents were added in non toxic

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concentrations for the cells. The solutions were stored in the dark at -20o C. Both of

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these probes are cell-permeable and are oxidized by the ROS mentioned above to

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DCF, that binds to DNA and emits green fluorescence, and ethidium bromide that

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binds to DNA and emits red fluorescence, respectively [33]. DCFH-DA (5 µΜ) and

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DHE (2 µΜ) were added to sperm samples and incubated in the dark at room

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temperature for 20 minutes. Apoptotic/dead spermatozoa were excluded by using

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counter nucleic acid stains. One µL (50 µg/mL) of Propidium Iodide (PΙ-Cat. 421301, 6

ACCEPTED MANUSCRIPT Biologend, London, UK) was added as a counterstain for DCFH-DA and a 5-minute

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incubation followed. YO-PRO 1 (Y3603, Invitrogen, Life Technologies, California,

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USA) was used as a counterstain for DHE. One µl of the YO-PRO 1 solution (10µM)

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was added in the sample and a 20-minute incubation followed. In the end of each

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incubation, 700 µl of Annexin V binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl,

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2.5 mM CaCl2) were added in each tube. The samples were centrifuged (300x g, 10

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min, 4ο C) and the pellet was resuspended with Annexin V binding buffer and

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analyzed by flow cytometry analysis within 10 minutes. The experiment was

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conducted 8 times.

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The detection of PS externalization in spermatozoa was accomplished by flow

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cytometry. Annexin V-FITC (SC 4252, Santa Cruz Biotechnology Inc., California,

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USA) is a protein which selectively binds to PS in a calcium-dependent manner and

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determines the accumulation of phosphatidylserine (PS) from the cytoplasmic

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interface to the extracellular surface. Following the manufacturer’s instructions, for

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each assay 1 x 105 washed spermatozoa (according to the technique described

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above for ROS determination) were diluted in 100 µl of Sperm Talp and 1 µl (50

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µg/250 µl) of Annexin V-FITC was added to the samples. The tubes were incubated

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for 15 minutes at RT in the dark. Propidium Iodide (PI) was used as a counterstain in

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order to exclude dead spermatozoa. After the addition of 700 µl of Annexin V binding

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buffer in each tube, the samples were centrifuged (300x g, 10 min, 4ο C) and the

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pellet was resuspended with Annexin V binding buffer and analyzed by flow

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cytometry analysis within 10 minutes. The experiment was conducted 8 times.

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2.2.4. Capacitation and Acrosome Reaction (AR)

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The sperm sample was prepared according to the method described in

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section 2.2.3. Spermatozoa were incubated either in the presence (positive control)

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or absence (negative control) of heparin (H0777), which is a well known capacitating

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factor or with different concentrations of crocin (0.5mM, 1mM, 2mM). After 4 hours of

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incubation [34], spermatozoa were exposed to 60 µg/ml lysophosphatidylcholine

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(L1381) for 15 minutes, which induces AR only in capacitated spermatozoa.

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Spermatozoa were first stained with Trypan blue (T6146) (in order to assess viability)

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and then smeared, dried and fixed in 37% formaldehyde with neutral red for 2 min.

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Afterwards, the dried smears were stained overnight with Giemsa (in order to assess

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the acrosome integrity). Smears were evaluated under microscopic examination

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(100x). The experiment was conducted 8 times.

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Lipid peroxidation was assessed on the basis of Malondialdehyde (MDA)

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formation. Malondialdehyde was determined by a selective third-order derivative

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spectrophotometric method (Shimadzu Model UV-160A, Burladingen, Germany),

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slightly modified for spermatozoa [35]. According to the method described in section

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2.2.2., 107 washed spermatozoa which have been previously treated or not with

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different concentration of crocin in a total volume of 50 µl (0.5mM, 1mM, 2mM) for 60

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and 180 min respectively, were mixed with 50 µl of FeSO4 7H2O (5mM) (Μerck,

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Germany) and 2900 µl of distilled water and further incubated for 60 min at 37o C.

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After incubation, the samples were mixed with 500 µl trichloroacetic acid 35%

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(Panreac, Spain) and 2000 µl butylated hydroxytoluene (W218405) in hexane and

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were centrifuged at 2000x g for 1 min. The top hexane layer was discarded and the

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bottom aqueous layer (2500 µl) was pipetted to another tube containing 1500 µl

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thiobarbituric acid (ΤΒΑ) 0.8% (T5500). After 30 min of incubation (70ο C), the tubes

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were allowed to cool under tap water, and submitted to third-order derivative

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spectrophotometry. The concentration of MDA (ng/ 107 spermatozoa) was calculated

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on the basis of the height of the third-order derivative peak at 521,5 nm by referring

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to slope and intercept data of the computed least squares fit of a standard calibration

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curve prepared using 1,1,3,3-tetrahethoxypropane. The experiment was conducted 8

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times.

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Bovine ovaries were obtained from a local abattoir and transported immediately

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within 2 h to the laboratory in warm saline (30-35o C) supplemented with kanamycin.

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Immature cumulus-oocyte complexes (COCs) were selected from 2-8 mm diameter

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follicles and aspirated with a scalp vein set equipped with a 21 gauge needle,

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connected to a vacuum pump (~ 40 mmHg). COCs were selected into a sterile conical

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tube containing TCM-Aspiration (TCM 199 with 25mM Hepes, 2mM sodium

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bicarbonate, 2mM pyruvic acid, 1mM L-glutamine, 10 µl/ml amphotericin B and 540

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µg/ml heparin supplemented with 2 % bovine serum) in 37o C thermal bath. The

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COCs were strictly evaluated and classified with standard criteria (at least a couple of

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layers of compact cumulus cells and an evenly granulated cytoplasm with no clear

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spaces). Retrieved COCs were washed properly twice in order to be cleaned of

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debris. The selected oocytes were placed in a 4-well plate containing TCM-IVM

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(TCM 199 supplemented with 15% bovine serum, 0.5 µg/ml FSH, 5 µg/ml LH, 0.8mM

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glutamine and 50 µg/ml gentamycin), were covered with 400 µl of mineral oil (M8410)

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in groups of twenty five oocytes/well and a 24 hour incubation (37o C, 5 % CO2 in air

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and saturated humidity) followed. Frozen bovine semen straws were used for in vitro fertilization. Straws were

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thawed in a water bath at 37o C for 40 sec. Motile spermatozoa were obtained with a

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80-45% Percoll gradient, using 2 ml of each one. Semen, layered on the top, was

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centrifuged (380x g, 10 min, RT). Two ml of Sperm Talp were used to wash the pellet

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(140x g, 10 min, RT). Sperm concentration was determined with a haemocytometer

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and adjusted on the concentration of 106 spermatozoa/ml with IVF Talp (114mM

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NaCl, 3.2mM KCl, 0.34mM NaH2PO4,, 0.5mM CaCl2, 10mM Na lactate, 10 mg/ml

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phenol red, 30 mg/ml heparin, 30µΜ penicillamine, 15µΜ hypotaurine, 1µM

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epinephrine supplemented with 10.4mM pyruvate, 50 µg/ml gentamycin and 1%

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bovine serum in water for embryo tranfer).

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Matured oocytes were recovered and transferred in 4-well plates containing IVF

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Talp medium in groups of twenty five oocytes/well. Afterwards, 10 µl of washed

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spermatozoa were transferred in each well and were covered with 400 µl of mineral

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oil. The most potent concentration of crocin (1mM), in terms of semen quality, was

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also added in each well up to a final volume of 400 µl in order to evaluate its impact

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on embryo production rate and blastocyst quality, in comparison with a control group.

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The gametes were incubated for 18 hours (37o C, 5 % CO2 in air and saturated

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humidity).

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Presumptive zygotes were stripped of cumulus cells by vortexing (2 min in TCM-

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Hepes+5 % bovine serum). The zygotes were retrieved with a mouth-pipette,

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transferred in IVC medium (SOF-medium supplemented with 30 µl/ml essential

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aminoacids, 10 µl/ml non essential aminoacids and 5% bovine serum) in groups of

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thirty zygotes/well were covered with 400 µl of mineral oil, and were incubated for 7

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days in a humidified mixture of 5% CO2, 7% O2, and 88% N2 in air, at a temperature of

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37o C. On day 7, embryo cleavage and blastocyst development were assessed in

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order to evaluate the effect of crocin on IVEP.

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Statistical analysis

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The data are presented as mean ± SD. The results were analyzed using SPSS

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(version 22.0, provided by the Aristotle University of Thessaloniki). Repeated

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measures ANOVA with the Bonferroni correction were used for the statistical

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analysis, where the interaction between different concentrations of crocin and the

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three time points was analyzed. The differences between groups in the mean values 9

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of IVEP were analyzed by t-test. A value of P<0.05 was considered statistically

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significant.

328 3. Results

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The results in Table 1 indicate a time-dependent effect of 1mM of crocin

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(P=0.001). The addition of crocin (1mM) resulted in improved maintenance of rapid

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spermatozoa after 120 min (P=0.035) and 240 min (P=0.007) of incubation,

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compared to the control group, while the same effect was observed in terms of total

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motility (rapid, medium, slow) after 120 min (P=0.032) and 240 min (P=0.042) of

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incubation, compared to the control group (Fig.1). Additionally, 0.5mM of crocin

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proved to be beneficial for the cells, because the incubation of spermatozoa with this

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concentration for 240 min resulted in a better maintenance of rapid movement

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(P=0.007) and total motility (P=0.037), compared to the control group. The other

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parameters (Progressive Motility, Medium, Slow, VCL, VSL, VAP and ALH) showed

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only a trend to increase (CASA parameters not shown), while the percentage of static

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spermatozoa showed a trend to decrease due to the presence of 1mM of crocin.

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The results of the viability assessment in Table 2 indicate that the percentage of

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alive spermatozoa with intact acrosome is influenced by the crocin concentration in a

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time-dependent manner (P=0.002). More specifically, 1mM of crocin resulted in a

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better maintenance of viability, compared to the control group, after 120 min (P=0.02)

346

and 240 min (P=0.001) of incubation. Statistical difference was observed between

347

the 1mM and the 2mM group (P=0.049) after 240 min of incubation. Furthermore, the

348

results indicate that there is no effect on the acrosomal integrity of spermatozoa due

349

to the presence of crocin under these incubation conditions.

EP

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While incubation time did not affect statistically DNA fragmentation (P=0.68), the

351

various concentrations of crocin had different effect on DNA integrity. Indeed,

352

spermatozoa treated with 1mM and 2mM of crocin showed statistically significant

353

lower DNA fragmentation index compared to the other groups (P<0.05, Table 3).

354

AC C

350

Figures 2 and 3 summarize the results from the evaluation of the intracellular

355

levels of ROS using flow cytometry. In general, there is an interaction between

356

concentration and time (P=0.042). Figure 2 reveals that, in comparison with the

357

untreated ones, the groups of 1mM and 2mM of crocin might scavenge and/or

358

prevent the production of superoxide anion after 120 min (P=0.001 and P=0.035,

359

respectively), while only the concentration of 1mM of crocin remained effective after

360

240 min of incubation (P=0.004). On the other hand, all the concentrations of crocin

361

significantly reduced the formation of hydrogen peroxide after 120 min of incubation,

362

and with a similar potency, compared to the control group (P<0.05). 10

ACCEPTED MANUSCRIPT 363

Figure 4 indicates that, after 240 min of incubation, the percentage of

364

spermatozoa with PS externalization was lower compared to the control group due to

365

the presence of 1mM (P=0.019) and 2mM (P=0.008) of crocin. Dot plot histograms

366

showing simultaneous measurements of PS externalization are presented in Figure

367

5. The histograms are representative of 8 different assays. The results from the evaluation of acrosomal status are presented in Table 3. The

369

acrosomal losses observed in 10% of the sperm population at time 0 min, can be

370

attributed to cryopreservation and freeze/thawing procedures. After 240 min of

371

incubation, sperm treatment both with heparin and 1mM of crocin resulted in a

372

significantly higher incidence of AR compared to the negative control (P<0.01). The

373

effect of 1mM of crocin is comparable, although statistical significant different, to that

374

of heparin.

SC

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368

In terms of lipid peroxidation, statistical analysis showed an interaction between

376

concentration and time (P<0.05). The results in Table 2 indicate that the presence of

377

1mM of crocin kept the MDA production of spermatozoa, after 120 and 240 min of

378

incubation, at very low levels, compared with any other group. Moreover, 0.5mM of

379

crocin protected spermatozoa from LPO only after 240min of incubation, compared to

380

the control and the 2mM group (P=0.001). It is noteworthy that the concentration of

381

1mM of crocin had the optimum effect on lipid peroxidation; the incubation of

382

spermatozoa with this concentration resulted to the production of almost half the

383

quantity of MDA that was detected in the 0.5mM group. Finally, it is obvious that the

384

highest concentration of crocin (2mM), resulted in loss of motility, viability and

385

increase of MDA production (Tables 1 and 2) . This concentration might diminish the

386

antioxidant potential of crocin.

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375

The percentages of cleavage and blastocyst rates are presented in Table 4. The

388

addition of 1mM crocin in the IVF media resulted in higher blastocyst’s crop

389

compared to the control group (P<0.01), while the two groups showed no statistically

390

significant difference in the percentage of cleavage rate.

391

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392

4. Discussion

393

Crocin is known for its antioxidant activity, both in vivo and in vitro [21,26]. In the

394

present study, the crocin concentrations were chosen following pre-experiments we

395

conducted, as well as according to the limited available data [27]. ROS and LPO

396

determination indicated that crocin (1mM) is a potent scavenger of both superoxide

397

anion and hydrogen peroxide, while this concentration successfully protected the 11

ACCEPTED MANUSCRIPT 398

phopsholipids of the plasma membrane from the oxidative attack of ROS. Crocin

399

scavenging of both hydroxyl radicals and superoxide anion is well established

400

[24,36]. However, our results indicate that crocin is also a potent scavenger of

401

hydrogen peroxide. Crocin successfully reduced the levels of both superoxide anion and hydrogen

403

peroxide. To date, more than 100 clinical and experimental studies have examined

404

the effect of antioxidants on sperm parameters [15]. However, Comhaire [37]

405

suggested that it is essential to conduct some laboratory trials such as ROS and

406

DNA fragmentation index determination in order to evaluate the effect of an

407

antioxidant agent on fertility. In the present experiment with, we conducted these

408

trials while we tried to examine the possible direct effect of crocin on spermatozoa

409

from many different aspects, as well.

SC

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402

The addition of crocin in the media proved to be beneficial for the cells in terms of

411

motility and viability at the concentration of 1mM. Gadea and co-authors [38]

412

proposed that the supplementation of the media with antioxidants, right after thawing,

413

blocks the production of ROS or counteracts oxygen toxicity. The stabilizing effect of

414

carotenoids on sperm preservation is associated with their interaction with

415

superoxide anion and not with singlet oxygen [24]. Moreover, crocin enhances the

416

activity of specific intracellular detoxifying enzymes or influences the strength and

417

fluidity of the membrane, thus affecting its permeability to oxygen and other

418

molecules [21].

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410

Motility is the most important indicator of the in vivo sperm fertilizing capacity [39].

420

Oxidative Stress predisposes deleterious effects on the fluidity, integrity and flexibility

421

of sperm plasma membrane, characteristics associated with fertilizing capacity [12].

422

Our results indicate that the presence of 1mM of crocin during bovine sperm

423

preparation had an advantageous effect on total motile and rapid spermatozoa

424

compared to the control group, while no influence on the other CASA kinematic

425

parameters was observed. The beneficial effect of saffron and its bioactive

426

constituent, crocin, on motility and viability has been proved in humans, in mice and

427

in red deer [22,23,25]. It is suggested that simple laboratory techniques such as the

428

motility assessment by CASA, the evaluation of the DNA fragmentation index and the

429

integrity of the plasma membrane are sufficient enough to predict field fertility [40].

430

Our results showed that crocin (1mM) preserved sperm motility, plasma membrane

431

integrity and kept DNA intact over time, probably through the modulation of MDA and

432

ROS concentration. In addition, the presence of 0.5mM of crocin proved to be

433

beneficial for the cells, especially for rapid and total motility after 240 min of

434

incubation. The maintenance of an appropriate ROS ratio is significant for adequate

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12

ACCEPTED MANUSCRIPT sperm functionality [41]. The fact that increased ROS levels have been correlated

436

with decreased sperm motility [42], provides a possible explanation why crocin at the

437

concentration of 2mM didn’t maintain the initial motility and viability of spermatozoa

438

with intact acrosome, compared to the control group. Furthermore, crocin at the

439

concentration of 2mM did not protect the PUFAs of the membrane from the

440

detrimental effect of ROS. It is most likely that low and controlled amounts of lipid

441

hydroxyperoxides (generated by PUFAs metabolism) are essential for the

442

maintenance of the membrane’s fluidity [9]. LPO can cause protein oxidation which

443

leads to motility loss. Therefore, the detection of high levels of MDA is negatively

444

correlated with the motility parameters [43] and the ability of spermatozoa to

445

penetrate the zona pellucida [44]. On the contrary, 0.5mM and 1mM of crocin proved

446

to be beneficial for the cells because spermatozoa were successfully protected from

447

LPO (Table 2). Besides it is known for many antioxidants, such as ascorbic acid, that

448

there is a beneficial maximum concentration, beyond which they act as pro-oxidants

449

and their presence in the media could be harmful for the cells.

M AN U

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435

Sperm DNA damage has been correlated with infertility, early pregnancy loss and

451

genetic abnormalities in the offspring [3]. Dobrinski and co-authors [45] reported that

452

the factors that may affect nuclear chromatin integrity in fresh bovine semen are

453

individuality and semen quality characteristics, as well as the variation between

454

ejaculates of the same bull [46,47]. On the other hand, DNA damage is often induced

455

by OS [7,42,48]. The percentage of fertilization and the embryo quality are lower

456

when spermatozoa produce high levels of ROS [7,49] while the addition of

457

antioxidants results in scavenging and/or reduction in the production of ROS [50] and

458

preserves sperm chromatin integrity [16]. Some DNA strand breaks can be repaired

459

by the oocyte just after fertilization. However, if the DNA damage is extensive,

460

apoptosis and embryo fragmentation may occur [51]. De Lamirande and Gagnon

461

suggested that hydrogen peroxide is responsible for DNA fragmentation and

462

abnormalities in chromatin integrity [52], while superoxide anion is also known to

463

cause nuclear DNA damage [10]. In our experiments, all concentrations of crocin

464

significantly suppressed the production of hydrogen peroxide after 120 min of

465

incubation, while the 1mM and 2mM concentrations resulted in a significantly lower

466

DNA fragmentation index. It is already established that crocin increases glutathione

467

peroxidase and superoxide dismutase activity, which detoxify ROS [26]. It is very

468

likely that the protective effect of crocin in the abovementioned concentrations is due

469

to scavenging or inactivation of hydrogen peroxide from cellular antioxidants.

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470

Acrosome reaction is a prerequisite for successful fertilization and is accompanied

471

with structural changes of the spermatozoon. It is believed that the lipid changes 13

ACCEPTED MANUSCRIPT (cholesterol efflux) that occur in the plasma membrane of sperm during capacitation

473

are related to the intrinsic membrane properties, such as permeability, adhesiveness

474

and fusibility [53]. A major contributor to the cholesterol efflux during capacitation is

475

OS. A study by O’ Flaherty and co-authors [54] examined the influence of ROS on

476

capacitation and the acrosome reaction in frozen/thawed bull sperm and they

477

concluded that ROS (especially superoxide anion) is required for the capacitation

478

process and may participate as an inductor of the acrosome reaction. Very low and

479

controlled concentrations of ROS (specifically superoxide anion, hydrogen peroxide

480

and nitric oxide) mediate the in vitro processes, either directly or indirectly, via the

481

activation of specific enzymes, such as kinases or phospholipase A2 (PLA2) [55]. The

482

data are converging to describe these events as oxidative or redox regulated [6]. The

483

incubation of human and bovine spermatozoa in capacitating conditions especially

484

stimulates the generation of superoxide anion [1, 55].The targets of ROS remain

485

unknown, but the tyrosine phosphorylation of specific proteins during capacitation

486

seems to be regulated by these molecules, especially hydrogen peroxide [55,56]. In

487

order to evaluate the effect of crocin on sperm capacitation and acrosome reaction,

488

we used three different concentrations of this antioxidant. We observed that crocin

489

modulated ROS concentration, and in the presence of 1mM of the antioxidant,

490

spermatozoa underwent capacitation and AR in percentages similar to heparin, a

491

well-known capacitating factor [34]. Finally, a direct effect of crocin on capacitation

492

should be taken under consideration. Carotenoids, such as lypopene and capsanthin,

493

induce cholesterol efflux [57,58] by the enhancement of PLA2. These modifications in

494

the architecture of plasma membrane increase the permeability to calcium ions and

495

bicarbonate and therefore protein tyrosine phosphorylation occurs. However, further

496

studies should be carried out in order to clarify the molecular events related to

497

capacitation after crocin’s supplementation.

SC

M AN U

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The phenomena of capacitation and AR are correlated with LPO. Mild peroxidative

AC C

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472

499

conditions improve the fertilizing potential of spermatozoa by increasing their binding

500

capacity to zona pelludica [9]. In addition, regarding ‘early apoptotic’ phenomena in

501

sperm such as PS externalization, our results with respect to sperm capacitation and

502

PS externalization underpin the proposal of Martin and co-authors [13] that there

503

might be a correlation between PS exposure in sperm membrane and AR. After 240

504

min of incubation with 1mM of crocin, spermatozoa, probably thanks to an optimum

505

level of ROS, suspended excessive LPO and modified PS externalization (30.52%),

506

resulting in capacitation as demonstrated by the induction of AR (32%) by LPC.

507

Interestingly, our data are in accordance with other authors who support that

508

cryopreservation and freeze/thawing procedures in bovine sperm trigger the 14

ACCEPTED MANUSCRIPT externalization of PS due to the destabilization of plasma membrane [11,59]. The

510

determination of PS externalization (Fig. 5) after thawing revealed a high proportion

511

of bull spermatozoa that express PS on their surface (Annexin+/PI-). Our results are

512

comparable to the findings of Anzar and co-authors (43.7% ± 4% vs 31 %) [11], but

513

not with Januskauskas and co-authors [59]. These discrepancies were attributed to

514

differences in semen samples and handling after thawing. Nevertheless, this

515

phenomenon is not accompanied with poor fertilization outcome (Table 4).

RI PT

509

Oxidative stress is also involved in the aetiology of defective embryo development

517

[7]. Bovine oocytes are capable of controlling the deleterious effects of ROS because

518

of their own enzymatic antioxidant activity, which is increased after in vitro maturation

519

[60]. Neverthelss, many antioxidants have been tried in vitro in order to improve the

520

maturation rates and the developmental competence of the oocytes [42]. However,

521

Dalvit et al. [61] showed that there was no difference in ROS production between

522

immature and matured oocytes. A significant increase in ROS levels in 2-cell

523

embryos was detected compared to the oocyte. A gradual increase in ROS

524

production was observed up to the late morula stage during IVC. This suggests that

525

oocyte maturation conditions are not responsible for OS. On the other hand, OS

526

contrived by male gametes has great significance in procedures involving ART [62].

527

In vitro incubation of oocytes with a critical number of ROS-producing spermatozoa

528

that remain outside the oocyte could lead to oxidative damage of the oocytes or

529

pronucleate embryos [63]. Spermatozoa are much more vulnerable to OS, which

530

compromises their fertilizing capacity. Since the pre-treatment of spermatozoa with

531

antioxidants before IVF prevents loss of motility and DNA fragmentation in the bull,

532

the addition of these compounds could be a very promising strategy to counteract the

533

negative effects of OS during IVF. To date, the beneficial effect of the pre-treatment

534

or supplementation during IVF with antioxidants remains controversial [16,28,29]. In

535

our study, the most effective concentration of crocin on thawed bovine sperm quality

536

proved to be 1mM and this concentration was also tested for the first time in bovine

537

IVF procedure, in terms of embryo cleavage and blastocyst rates. Indeed so, the

538

addition of 1mM of crocin in the media of in vitro fertilization resulted in a significantly

539

higher blastocyst production (P<0.01) compared to the control group (Day 7). We

540

attribute this result to the modulation of ROS concentration by crocin; nevertheless a

541

direct effect of crocin on the fertilizing capacity of spermatozoa should not be

542

excluded. In any case, it is impossible to dissect the effect of crocin on spermatozoa

543

and/or oocytes/zygotes.

544

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516

Conclusion

15

ACCEPTED MANUSCRIPT Taking into account that in sperm incubated with crocin the levels of intracellular

546

ROS were lowered, the generation of MDA was suppressed, and the percentage of

547

capacitated and acrosome reacted spermatozoa was increased, we suggest that

548

crocin ensures controlled amounts of ROS and lipid hydroxyperoxides, thus

549

improving sperm fertilizing capacity and fertilization outcome. The latter was verified

550

by the significantly higher blastocyst rate in IVF procedure. Further studies should be

551

conducted in order to clarify the molecular mechanism of crocin’s action, the potential

552

in vivo dose-dependent effect on fertilization procedure and the effect of crocin on

553

oocytes/zygotes.

RI PT

545

554 Acknowledgements

556

This study has been supported by a grand of the Research Committee of Aristotle

557

University, Thessaloniki, Greece.

558

This paper is dedicated to the memory of our colleague, Prof. Zaphiris Abas, who

559

unexpectedly passed away. We also wish to thank Dr P. Kotandaki and Mrs Ch.

560

Bekiari for their unstinting contribution to our experiments.

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555

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19

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[48] Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z, Irvine DS.

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Relative impact of oxidative stress on the functional competence and genomic

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integrity of human spermatozoa. Biol Reprod 1998;59:1037-46.

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[49] Aitken RJ, Irvine DS, Wu FC. Prospective analysis of sperm-oocyte fusion and

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reactive oxygen species generation as criteria for diagnosis of infertility. Am J Obstet

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Gynecol 1991;164:542-51.

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[50] Lopes AS, Lane M, Thompson JG. Oxygen consumption and ROS production

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are increased at the time of fertilization and cell cleavage in bovine zygotes. Hum

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Reprod 2010;25:2762-3.

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[51] Agarwal A,Allamaneni S. Oxidative stress and Human Reproduction. In: Singh

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KK editor, Oxidative stress, disease and cancer, USA, Imperial College Press Co;

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2006: p 687-703.

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[52] de Lamirande E, Gagnon C. Reactive oxygen species and human spermatozoa:

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effects on the motility of intact spermatozoa and sperm axonemes. J

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1992;13:368-78.

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[53] Gordon I. In vitro Fertilization In: Gordon I editor, Laboratory production of cattle

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embryos. edn 2, Wallingford, UK, CABI Publishing; 2003: p 176-219.

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[54] O’ Flaherty CM, Beorlequi NB, Beconi MT. Reactive Oxygen species

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requirements for bovine sperm capacitaion and acrosome reaction. Theriogenol

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1999;52:289-301.

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[55] Rivlin J, Mendel J, Rubinstein S, Etkovitz E, Breitbart H. Role of Hydrogen

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Peroxide in Sperm Capacitation and Acrosome Reaction. Biol Reprod 2004;70:518-

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[56] Visconti PE. Understanding the molecular basis of sperm capacitation through

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kinase design. PNAS 2009;106:667-8.

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[57] Palozza P, Simone R, Catalano A, Parrone N, Monego G, Ranelletti FO.

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Lycopene regulation of cholesterol synthesis and efflux in human macrophages. J

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[58] Aizawa K, Inakamura T. Dietary capsanthin, the main carotenoid of paprika

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(Capsicum annuum), alters plasma high-density lipoprotein-cholesterol levels and

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hepatic gene expression in rats. Br J Nutr 2009;102:1760-6.

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[59] Januskauskas A, Johannisson A, Rodriguez-Martinez H. Subtle membrane

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changes in cryopreserved bull semen in relation with sperm viability, chromatin

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structure, and field fertility. Theriogenol 2003;60:743-58.

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690

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P, Ménézo, Y. Expression of genes encoding

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antioxidant enzymes in human and mouse during the final stages of maturation. Mol

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Hum Reprod 1999;5:720-5.

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[61] Dalvit GC, Cetica PD, Pintos LN, Beconi MT. Reactive oxygen species in bovine

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embryo in vitro production. Biocell 2005;29:209-12.

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[62] Baker MA, Aitken RJ. Reactive Oxygen Species in spermatozoa: methods for

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monitoring and significance for the origins of genetic disease and infertility. Reprod

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Biol Endocrinol 2005;3:67.

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[63] Alvarez JG, Minaretzis D, Barrett CB, Mortola JF, Thompson IE. The sperm

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stress test: a novel test that predicts pregnancy in assisted reproductive

736

technologies. Fertil Steril 1996;2:400-5.

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737

Fig.1. The effect of 3 different concentrations of crocin on the percentage of total

739

motility of spermatozoa. Data are presented as mean ± SD. Different letters (a,b)

740

indicate statistically significant differences between the different concentrations of

741

crocin within each given time point (P<0.05, n=8).

742

Fig.2 The effect of 3 different concentrations of crocin οn the production of

743

superoxide anion. Data are presented as mean ± SD. Different letters (a,b,c) indicate

744

statistically significant differences between the different concentrations of crocin

745

within each given time point (P<0.05, n=8).

746

Fig.3 The effect of 3 different concentrations of crocin οn the production of hydrogen

747

peroxide. Data are presented as mean ± SD. Different letters (a,b) indicate

748

statistically significant differences between the different concentrations within each

749

given time point (P<0.05, n=8).

750

Fig.4. The effect of 3 different concentrations of crocin οn the percentage of

751

spermatozoa with PS externalization. Data are presented as mean ± SD. Different

752

letters (a,b,c) indicate statistically significant differences between the different

753

concentrations within each given time point (P<0.05, n=8).

754

Fig.5. Dot plot histograms representing simultaneous measurements of PS

755

externalization of bovine spermatozoa (1 x 105) during 240 min of incubation. The

756

histograms are representative of different assays.

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Table 1. The effect of three different concentrations of crocin on motility parameters

120

Medium

Slow

Static

Progressive

(%)

(%)

(%)

(%)

Motile (%)

Control

47.73±8.51

15.26±6.43

2.28±2.55

34.73±3.54

28.65±13.38

0.5 mM

44.21±3.95

17.06±5.29

2.22±1.31

36.51±5.69

19.77±5.38

1 mM

46.87±4.80

12.07±4.95

3.98±2.24

37.08±4.65

19.56±4.82

2 mM

47.30±4.82

13.71±6.79

3.63±2.43

35.36±2.97

18.57±1.85

22.65±9.95

b

14.98±6.85

9.37±4.88

53.00±11.69

20.52±11.12

29.5±10.12

b

13.91±6.41

5.82±3.23

50.77±8.85

24.03±10.29

43.07±9.40

a

10.22±6.96

5.67±5.57

41.04±13.96

24.81±9.97

28.61±15.15

14.56±7.29

5.70±5.96

51.13±13.37

17.87±8.41

13.87±8.22

b

10.66±8.94

4.15±2.30

71.32±22.96

24.45±19.98

28.72±7.33

a

Control 0.5 mM 1 mM 2 mM

240

Control 0.5 mM 1 mM

9.22±8.21

7.98±4.50

54.08±13.81

22.28±9.80

a

14.40±9.70

10.20±7.40

54.43±17.59

15.76±8.10

ab

11.97±11.47

9.29±5.85

59.08±17.13

18.83±12.70

31.27±12.42 19.66±17.67

EP

2 mM

Values with different superscripts indicate statistical difference between the treatments in each given time point (P<0.05)

AC C

a,b

b

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Rapid

M AN U

0

Treatment

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Τime

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(mean ± SD) of bovine spermatozoa (n=4, 8 replicates)

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Table 2. Alive spermatozoa (%) with intact acrosome and MDA (ng/107 spermatozoa) production (mean ± SD) of samples supplemented in vitro

Alive spermatozoa with intact acrosome (%) Control

0

49.87±6.25

120

34.87±8.65

b

240

19.00±9.78

b

1mM

48.25±7.1

2mM

47.87±9.76 b

38.12±10.5

26.12±10.26

b

Control

50.12±6.17 a

38.5±7.32

a

25.74±8.24

38.75±8.95

7.1±1.6

b

46.76±10.55

0.5mM

6.2±1.5

203.5±58.5 b

1mM

a

234.2±49.5

a

158.8±35

a

b

93.9±42.4

2mM

5.3±1.5

6.4±1.8 b

230.9±33.3

a

b

175.4±43.1

a

99.8±48.5

41.7±23.9

M AN U

a,b

0.5mM

7

MDA production (ng/10 spermatozoa)

SC

Time

RI PT

with three different concentrations of crocin (n=4, 8 replicates)

Values with different superscripts indicate statistical difference between the treatments in each given time point (P<0.05)

Table 3. DNA fragmentation index and acrosomal status (mean ± SD) of bovine spermatozoa supplemented in vitro with three different

Alive spermatozoa with Acrosome Reaction

a,b,c,d

9.13 ± 0.42 d

10±2.3

a

7.25 ± 0.42 c

19±3.9

ab

1mM (%)

2mM (%) b

5.81± 0.42 b

32±2.7

6.63 ± 0.42

Control +Heparin (%) b

c

15±2.9

AC C

Spermatozoa with fragmented DNA

0.5mM (%)

EP

Control (%)

TE D

concentrations of crocin (n=4, 8 replicates)

a

39±4.4

Values with different superscripts indicate statistical difference between the treatments in each experiment(P<0.05).

23

ACCEPTED MANUSCRIPT Table 4. The effect of 1mM of crocin on cleavage rate and blastocyst development (mean ± SD) compared with the control group (P<0.05, n=8 replicates) Cleaved

Cleavage

BL

rate

(N)

(N)

(N)

Control

293

246

110

83.9±7.5

1mM

393

297

213

75.5±11.9

Embryo

Embryo

production

production

(%)

(%)

1

(%)

44.7±8.45

37.5±8.45

71.7±9.7 *

54.2±9.7 *

Asterisks signify statistically significant differences between the groups (P<0.01).

1

Referred to the total number of cleaved oocytes, Referred to the total number of COCs

M AN U

used in IVC

SC

*

2

2

RI PT

COCs

AC C

EP

TE D

Abbreviations: COCs=Cumulus Oocytes Complexes

24

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