Freezing, Vitrification, and Freeze-Drying of Equine Spermatozoa: Impact on Mitochondrial Membrane Potential, Lipid Peroxidation, and DNA Integrity

Accepted Manuscript Freezing, Vitrification And Freeze-Drying Of Equine Spermatozoa: Impact On Mitochondrial Membrane Potential, Lipid Peroxidation And Dna Integrity Giovanni Restrepo, Elizabeth Varela, Juan Esteban Duque, Jorge Enrique Gómez, Mauricio Rojas PII:

S0737-0806(18)30565-3

DOI:

10.1016/j.jevs.2018.10.006

Reference:

YJEVS 2600

To appear in:

Journal of Equine Veterinary Science

Received Date: 30 July 2018 Revised Date:

2 October 2018

Accepted Date: 5 October 2018

Please cite this article as: Restrepo G, Varela E, Duque JE, Gómez JE, Rojas M, Freezing, Vitrification And Freeze-Drying Of Equine Spermatozoa: Impact On Mitochondrial Membrane Potential, Lipid Peroxidation And Dna Integrity, Journal of Equine Veterinary Science (2018), doi: https:// doi.org/10.1016/j.jevs.2018.10.006. 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.

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FREEZING, VITRIFICATION AND FREEZE-DRYING OF EQUINE SPERMATOZOA: IMPACT ON

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MITOCHONDRIAL MEMBRANE POTENTIAL, LIPID PEROXIDATION AND DNA INTEGRITY

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Giovanni Restrepoa*, Elizabeth Varelab, Juan Esteban Duqueb, Jorge Enrique Gómezb,

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Mauricio Rojasc a

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Faculty of Agricultural Sciences, Universidad Nacional de Colombia, Medellín, Colombia.

Faculty of Agricultural Sciences, Politécnico Colombiano Jaime Isaza Cadavid, Medellín, Colombia. c

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Institute of Medical Research, Universidad de Antioquia, Medellín, Colombia.

*Corresponding author: Giovanni Restrepo, Faculty of Agricultural Sciences, Universidad Nacional de

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Colombia - Sede Medellín, Cra 65 # 59A-110, Medellín, Colombia. E-mail address:

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[email protected]

12 ABSTRACT

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Maintaining the integrity of equine sperm subjected to preservation protocols is essential for the

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successful development of assisted reproduction procedures. The aim of this study was to assess the

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mitochondrial membrane potential, lipid peroxidation and DNA integrity of equine sperm subjected to

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freezing, vitrification and freeze-drying. Eight ejaculates obtained from four Colombian Creole horses

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were subjected to programmable freezing, vitrification and freeze-drying. After thawing or

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rehydration, sperm motility and kinetics were assessed through a CASA system. The mitochondrial

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membrane potential (∆ΨM), lipid peroxidation (LPO) and DNA fragmentation index (DFI) of the

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spermatozoa were assessed by flow cytometry using the DiOC6 (3), C11-Bodipy 581/591 and

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propidium iodide (PI) fluorescent dyes. The statistical analysis was conducted via generalized linear

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models (GLM), mean comparisons via the Duncan test and a principal component analysis. A higher

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rate of spermatozoa with a high-∆ΨM was found for freeze-drying (40.26 ± 7.79%) compared to

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freezing (21.82 ± 5.38%) and vitrification (5.32 ± 1.17%) (p <0.05). Likewise, a higher rate of non-

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peroxidized viable spermatozoa (Bodipy- / PI-) was found for freeze-drying (35.98 ± 7.01%) in

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relation to frozen (10.34 ± 2.69%) and vitrified (7.07 ± 2.00%) sperm (p <0.05). The DFI of vitrified

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spermatozoa (0.12 ± 0.04%) was higher when compared with the frozen (0.03 ± 0.01%) and freeze-

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dried (0.02 ± 0.01%) samples (p <0.05). The researchers conclude that vitrification generates greater

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sperm alterations than freeze-drying and freezing, while freeze-drying produces lower LPO and higher

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∆ΨM for equine spermatozoa.

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Keywords: cryopreservation, equine, freeze-drying, sperm, vitrification.

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1. Introduction

3 There are a number of reproduction techniques having a positive impact on the equine breeding

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industry [1, 2]. As a technology supplementing the various assisted reproduction processes, equine

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semen cryopreservation is an important tool for improving the genetics of a species by maximizing the

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use of good breeding [3], since it allows for the transportation and long term storage of semen [4].

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There are several methodologies for semen preservation and cryopreservation whose variations lie

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chiefly in the storage methods [5], packaging systems [6], cooling rates [7], media components [8],

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cryoprotectants employed and their concentration [9], and technical resources used [10]. The success

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of equine semen freezing has been directly associated with the individual effect of the breed on sperm

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cryotolerance [11] and with the decrease in the number of alterations affecting total and progressive

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motility, curvilinear velocity, linearity, plasma membrane permeability, morphology, DNA

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fragmentation, lipid peroxidation and even sperm mitochondrial activity [12-15]. On the other hand,

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equine sperm vitrification has shown low ability to preserve motility and a significant reduction of

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viability and acrosome integrity [16]. Another important disadvantage of semen cryopreservation

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methods is the need to use liquid nitrogen tanks, which have been associated with issues such as

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constant maintenance, possible viral contamination of the straws and the potential loss of sperm

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quality due to unexpected delays [17, 18]. A recent alternative is freeze-drying, i.e. the process of

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removing most of the water from a sample under vacuum and low temperature conditions [19].

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Freeze-drying has been used for sperm preservation with different species such as bovines [20] and

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pigs [21], for which blastocyst production has been reached. Additionally, the birth of live offspring

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has been reported for mouse [22], rats [23], rabbits [24] and equines [18]. This has been possible

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whenever sperm is injected into the oocytes using ICSI without requiring them to be motile. In

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addition, liquid nitrogen is not required to store freeze-dried semen, and the samples for use in ICSI

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may be packaged with very few spermatozoa per dose. This in turn makes it possible to prepare a high

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number of aliquots from one ejaculate [18]. Further advantages include that freeze-dried sperm can be

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temporarily stored at room temperature without requiring specialized cryoprotectants (25). However,

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like freezing and vitrification, preserving sperm via freeze-drying also generates alterations which may

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affect the viability of such cells. It is known that, during freeze-drying, the DNA of equine sperm may

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be damaged by freezing stress, drying or rehydration [26]. In addition, the impact of freeze-drying on

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the mitochondrial integrity of buffalo bull spermatozoa has been assessed [27]. Nevertheless, there is

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limited information on the effect of freeze-drying on mitochondria and other structural components of

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spermatozoa. This study aimed at assessing the mitochondrial membrane potential (∆ΨM), lipid

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peroxidation (LPO) and the DNA fragmentation index (DFI) of equine sperm subjected to freezing,

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vitrification and freeze-drying.

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2. Materials and methods

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2.1 Semen collection and assessment

6 The study included four Colombian Creole stallions (Equus caballus) located in the Aburrá valley, in

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the department of Antioquia (Colombia). The animals were active breeding stallions whose age ranged

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from 5 to 8 years. Likewise, their fertility had been confirmed by their live offspring, and their

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handling and stabling conditions were similar. Finally, their body condition scores ranged from 7 to 8

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(using a scale from 1 to 9). Samples were collected using a Missouri model artificial vagina (Minitube,

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Germany) previously lubricated with non-spermicidal gel. Two ejaculates were collected from each

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horse with a difference of one week, and each ejaculate was processed separately. A mare was used in

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order to increase sexual stimulation. The gel fraction of the ejaculate was removed through filtration,

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and the sperm was diluted at a ratio of 1:1 using EquiPlus® extender (Minitube, Germany) at a

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temperature of 32 ºC. This study included only those samples whose total motility was 60% or higher;

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this parameter was assessed with an optical microscope (Eclipse E200, Nikon, Japan). Before

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processing, the sperm was transported for two hours at 5 ºC in an Equitainer® (Hamilton Research

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Inc., USA).

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2.2 Spermatozoa freezing, vitrification and freeze-drying

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Each ejaculate was split into three aliquots which were randomly assigned to one of three preservation

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methods, namely: freezing, vitrification or freeze-drying. For the freezing process, the sperm was first

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subjected to centrifugation at 800 g for 10 min (Mikro 220R, Hettich, Germany). Then the pellet was

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resuspended using EquiPlus® extender (Minitube, Germany) supplemented with 5% centrifuged egg

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yolk (CEY) and 5% N,N-Dimethylformamide (Sigma-Aldrich, USA) until reaching a concentration of

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100 x 106 spermatozoa mL-1. The CEY was obtained as per the recommendations of Montoya et al.

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[28]. The sperm suspension was packaged in 0.5 mL straws (MRS1 Dual V2, IMV Technologies,

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France), which were refrigerated at 5 °C for 30 min and then transferred to the cryo-chamber of a

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programmable freezer (Cryocontroller PTC-9500, Crysalys, USA), where they were submitted to

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temperature decrease rates of -8 °C min-1 between 5 °C and -6 °C, and of -0.6 °C min-1 between -6 °C

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and -32 °C. Finally, the straws were stored in liquid nitrogen.

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Vitrification was performed as per aversion of the protocol described by Isachenko et al. [10] modified

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for equine semen. The sperm underwent centrifugation at 800 g for 10 min (Mikro 220R, Hettich,

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Germany). The pellet was resuspended using EquiPlus®extender (Minitube, Germany) supplemented 3

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with 5% bovine serum albumin (BSA, Sigma-Aldrich, USA) and 0.25 M of sucrose (Sigma-Aldrich,

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USA), until reaching a concentration of 100 x 106 spermatozoa mL-1. The sperm suspension was

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packaged in 0.5 mL straws (MRS1 Dual V2, IMV Technologies, France), which were then

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horizontally submerged in liquid nitrogen for 30 sec and stored in that same medium.

5 Freeze-drying was performed via a modified protocol described by Choi et al. [18]. The sperm

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underwent centrifugation at 800 g for 15 min (Mikro 220R, Hettich, Germany). The pellet was

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resuspended in a medium composed of DMEM/F-12 (Sigma–Aldrich, USA) supplemented with 10%

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bovine fetal serum (Invitrogen, USA) until reaching a concentration of 500.000 spermatozoa mL-1.

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The sperm suspension was packaged in cryovials of 2 mL that were then transferred to a freezer and

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kept there for 24 hours at -20 °C. Then, the samples were transferred to the pre-cooled chamber of a

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freeze-dryer (FreeZone 1 lt, Labconco, USA) and subjected to a 48 h cycle at a condensation

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temperature of -50 °C and a vacuum pressure of 150 mTorr. The cryovials were kept at room

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temperature in vacuum-sealed bags.

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2.3 Assessment of sperm motility

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After a storage period of one month, the straws with frozen and vitrified sperm were heated in water at

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37 °C for 60 sec, whereas the freeze-dried sperm was rehydrated by adding 500 µl of phosphate buffer

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solution (PBS) per vial. For each ejaculate, two samples of spermatozoa submitted to each treatment

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were evaluated. Sperm motility was assessed with the Sperm Class Analyzer® computer system

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(Microptic S.L, Spain), an Eclipse E200 phase contrast microscope (Nikon, Inc., Japan) and a Scout

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SCA780 digital camera (Basler, USA). This study assessed the total motility (TM), progressive

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motility (PM), curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity

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(VAP) of the spermatozoa.

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2.4 Analysis through flow cytometry

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The mitochondrial membrane potential (∆ΨM) of the spermatozoa was assessed with an adapted

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version of the protocols described by Rojas et al. [29] and Marchetti et al. [30]. A polystyrene tube

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was used to deposit 3.3'-dihexyloxacarbocyanine iodide (DiOC6, Molecular Probes, USA) in PBS at a

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final concentration of 80 nM and 7-aminoactinomycin D (7-AAD, Molecular Probes, USA) at a final

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concentration of 2 µg mL-1. The compounds were pipetted and then distributed equally in three tubes.

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Then, between 10 and 20 µL of frozen, vitrified or freeze-dried sperm were added. Subsequently, in

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order to stain the cells to simultaneously evaluate their viability, 1 µg mL-1 of propidium iodide (PI,

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Live/Dead® Sperm Viability Kit, Molecular Probes, USA) was added to each tube. The samples were

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incubated for 30 min, and the ∆ΨM was measured by flow cytometry (LSRFortessa™, BD 4

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Biosciences, USA). The samples were excited using a 488 nm solid phase laser and fluorescence from

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DiOC6 and 7-AAD was detected at 530/30 nm and 630/30 nm, respectively.

3 Sperm LPO was assessed through a modified version of the protocol reported by Ortega et al. [31]. In

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a tube 12 µL of C11-Bodipy 581/591 (Molecular Probes, USA) and 30 µL of 7-AAD (Molecular

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Probes, USA) were poured. Subsequently, the compounds were pipetted and the content was

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distributed equally in three tubes. Then, between 10 and 20 µL of frozen, vitrified or freeze-dried

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sperm were added. Finally, in order to stain the cells to simultaneously evaluate their viability, 1 µg

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mL-1 of PI was added to each tube (Molecular Probes, USA). The samples were incubated for 30 min

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at 37 °C and the degree of peroxidation was measured via flow cytometry (LSRFortessa™, BD

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Biosciences, USA).

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Sperm DNA fragmentation was assessed through PI staining, as described by Darzynkiewicz et al.

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[32]. Cells were fixed with 300 µL of ethanol at 70% (Merck, Germany) prepared in PBS (pH 7.4), for

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12 h at 4 °C. Samples were centrifuged (500 g, 5 min, 4 °C) and the resulting pellets were washed

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twice more with 3.0 mL of PBS. Cells were stained with PI 1 µg mL-1 in PBS with EDTA 0.37% p/v

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and 0.01% v/v of Triton X-100 and 200 U mL-1 of RNase A (Sigma, Aldrich). After 30 min of

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incubation in the dark at room temperature, the data were collected with a flow cytometer

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(LSRFortessa™, BD Biosciences, USA). Flow cytometry data from all tests were analyzed using the

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FlowJo version 7.6.2 (FlowJo, LLC, USA) software.

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2.5 Statistical Analysis

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The statistical analysis was conducted by adjusting generalized linear models (GLM) for DFI, ∆ΨM

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and LPO. For analyzes of ∆ΨM and LPO different sperm populations were established according to

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their viability, which was evaluated with PI. The fixed effects of stallion, ejaculate and treatment were

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included. Each of the two ejaculated from the same stallion was taken as a replicate. Ten thousand

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events (spermatozoa) were evaluated per sample of each treatment from each ejaculate. Data normality

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was assessed for each variable using the Kolmogorov-Smirnov test, and means were compared via the

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Duncan test. Results were expressed as mean ± standard error of the mean. The statistical analysis was

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carried out using the SAS 9.2 (SAS Inst. Inc., Cary, NC, USA) software. In addition, an analysis of

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principal components was conducted that took into account the preservation methods and the DFI,

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∆ΨM and LPO variables. Such analysis was performed using the StatGraphics Centurion XVITM

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(Statgraphics Technologies, Inc., USA) software.

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3. Results and Discussion

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This study had fresh samples of equine semen whose sperm motility and kinetics related features were

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consistent with those reported for Colombian Creole horses in previous studies [33, 34]. Table 1

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shows the results for the sperm motility and kinetics parameters for the fresh, frozen, vitrified and

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freeze-dried samples. Post-thawing results showed a marked decrease in sperm motility consistent

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with the findings reported by other studies using programmable freezing [35, 36].

6 Sperm vitrification has been relatively successful for species such as humans [37], canines [38], ovines

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[39] and rabbits [40]. However, it generally results in a strong decrease in motility. In this study, a

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decreased ratio of motile spermatozoa was observed after post-vitrification thawing along with a

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serious deleterious effect on kinetic parameters (see Table 1). A recent study reports low rates of

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equine motile spermatozoa after vitrification (<10%) when using diluents supplemented with 1% BSA

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and the lowest concentrations of sucrose (0.15 M and 0.3 M) and trehalose (0.15 M) [18], which is

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consistent with the observations in the present study, where a combination of 5% BSA and 0.25 M of

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sucrose was used. Hidalgo et al. [41] found higher values of motility and kinetics of vitrified stallion

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spermatozoa in media with different concentrations of sucrose (20 mM, 50 mM and 100 mM) or BSA

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(1%, 5% and 10%). However, unlike our research, the sperm suspensions were vitrified after 5 min of

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equilibration at room temperature (≈ 22 °C) or after 2 h of cold-storage (5 °C). Additionally, our

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sucrose concentration was higher (equivalent to 250 mM), which could have generated deleterious

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osmotic effects. Previous reports show that equine sperm are apparently more sensitive to chemical or

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osmotic toxicity generated by sucrose concentrations than the sperm from other species can tolerate

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[10, 38, 42].

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Cryoprotectant-free vitrification has become such a rapid and promising alternative to conventional

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methods resulting in post-thaw good-quality spermatozoa [37, 43], because classical vitrification

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requires a high proportion of permeable cryoprotectants in the medium and seems to be unsuitable for

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the vitrification of spermatozoa, due to the lethal osmotic effects and possible chemical alterations

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[44]. Alternative methods of sperm vitrification with high concentrations of cryoprotectants and

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previous exposure of semen packed in cryovials to liquid nitrogen vapors have been evaluated [45].

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Although this method was carried out in a previous study by our research team with better motility

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results than those found in this research [46], this methodology could be more similar to a rapid

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freezing process.

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No motility was observed for the freeze-dried spermatozoa after rehydration (see Table 1), which may

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be considered as an expected finding since, with the exception of an early study reporting a high rate

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of freeze-dried equine spermatozoa that recovered their motility upon rehydration [47], freeze-dried

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mammal spermatozoa normally lose motility, thus becoming unable to fertilize oocytes both in vivo

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and in vitro [25]. However, injection into an oocyte via ICSI does not require spermatozoa to be

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motile, since there are reports of live offspring from equine freeze-dried spermatozoa [18].

3 Table 1.

Motility and kinetics of fresh, frozen, vitrified and freeze-dried spermatozoa.

Treatment

TM (%)

PM (%)

VCL (µm/s)

VSL (µm/s)

VAP (µm/s)

Fresh

73.72 ± 5.19

41.22 ± 4.49

91.30 ± 5.04

34.67 ± 3.38

62.05 ± 4.51

Freezing

37.25 ± 4.34

19.46 ± 1.39

77.88 ± 5.62

Vitrification

4.31 ± 1.17

0.19 ± 0.08

20.96 ± 3.43

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Freeze-drying

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38.40 ± 4.93

53.30 ± 4.17

4.89 ± 1.29

9.81 ± 1.75

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Freezing: programmable freezing in a extender supplemented with 5% centrifuged egg yolk and 5%

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N,N-Dimethylformamide. Vitrification: direct immersion in liquid nitrogen in an extender

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supplemented with 5% of bovine serum albumin and 0.25 M of sucrose. Freeze-drying: 48 h freeze-

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drying cycle (condensation -50 °C, vacuum pressure 150 mTorr) in a medium composed of DMEM/F-

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12 and 10% of bovine fetal serum. TM: total motility. PM: progressive motility. VCL: curvilinear

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velocity VSL: straight line velocity. VAP: average path velocity. Results are shown as mean ±

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standard error of the mean.

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A factor that could influence the results of sperm quality is the sperm concentration after dilution.

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Findings revealed that treatments in which semen was diluted to a low concentration had lower initial

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total motility, progressive motility and plasma membrane integrity, a phenomenon known as the

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“dilution effect” of semen [48]. It has also been reported that sperm concentrations during freezing

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affect post-thaw semen quality of ejaculates, reducing the number of sperm that survives both the

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procedure and its functionality (lower motility and active mitochondria) [49].

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Mitochondrial membrane potential (∆ΨM) has been used as a measure of mitochondrial function and

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is linked to a host of mitochondrial functions, including ATP synthesis, import of mitochondrial

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proteins, calcium homeostasis and metabolite transport [50]. Table 2 shows the results of assessing the

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∆ΨM of the sperm subjected to the studied preservation methods. Statistically significant differences

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were found among the spermatozoa populations for the three preservation methods regarding ∆ΨM (p

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<0.05). Various studies have described the decrease in the ∆ΨM of frozen sperm as being associated

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with factors such as ejaculate freezability [51] or stallion age [52]. In contrast, no other reports were

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found that assess the effect of vitrification or freeze-drying on the mitochondrial activity of equine

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sperm. Studies on human and canine semen vitrification achieved the maximum level of ∆ΨM

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maintenance after vitrification using sperm that had been prepared in media consisting of 1% albumin

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(either bovine serum albumin or human serum albumin, respectively) and 0.25 M of sucrose [38, 53].

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ACCEPTED MANUSCRIPT 1 On the other hand, a similar protection has been reported for the mitochondria of bovine spermatozoa

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which underwent freeze-drying in media with and without trehalose and ethylene glycol-bis (2-

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aminoethylether) -N,N,N′,N′-tetraacetic acid (EGTA) [54]. In the present study, vitrification caused

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maximum decrease in the populations of spermatozoa with high and medium ∆ΨM. Likewise, it

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generated the highest rate of non-viable spermatozoa (PI+). The alterations observed in equine sperm

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subjected to high freezing rates like those used for vitrification have been attributed mainly to the

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osmotic shock experienced during thawing [55]. In contrast, freeze-drying was more efficient for

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preserving those spermatozoa with high-∆ΨM, and had a lower ratio of non-viable cells (PI+). A

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recent study also showed the ability of freeze-drying to preserve mitochondria. This was done by

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conducting an ultrastructural assessment of buffalo sperm [27].

Table 2. Mitochondrial membrane potential (∆ΨM) of frozen, vitrified and freeze-dried equine sperm. Treatment

High-∆ΨM (%)

Medium-∆ΨM

Low-∆ΨM (%)

PI+ (%)

9.13 ± 2.46a

2.29 ± 0.80b

66.41 ± 2.69b

5.22 ± 3.76a

9.91 ± 10.25a

88.03 ± 4.63a

10.66 ± 4.17a

2.48 ± 0.81b

44.17 ± 8.48c

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(%)

Freezing

21.82 ± 5.38b

Vitrification

5.32 ± 1.17c

Freeze-drying

40.26 ± 7.79a

Freezing: programmable freezing in a extender supplemented with 5% centrifuged egg yolk and 5%

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N,N-Dimethylformamide. Vitrification: direct immersion in liquid nitrogen in an extender

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supplemented with 5% of bovine serum albumin and 0.25 M of sucrose. Freeze-drying: 48 h freeze-

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drying cycle (condensation -50 °C, vacuum pressure 150 mTorr) in a medium composed of DMEM/F-

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12 and 10% of bovine fetal serum. High-∆ΨM: spermatozoa with high mitochondrial membrane

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potential. Medium-∆ΨM: spermatozoa with medium mitochondrial membrane potential. Low-∆ΨM:

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spermatozoa with low mitochondrial membrane potential. PI+: non-viable spermatozoa. Results are

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shown as mean ± standard error of the mean. Different letters indicate statistically significant

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differences (p <0.05).

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LPO has been associated with an increase in the membrane permeability and the loss of capacity to

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regulate the intracellular concentrations of ions involved in the control of sperm movement [50]. The

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assessment of LPO levels (see Table 3) showed differences between the population of non-peroxidized

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viable spermatozoa (Bodipy- / PI-) and that of peroxidized non-viable spermatozoa (Bodipy+ / PI+) (p

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<0.05), the latter being the most representative for the various preservation methods. Freeze-drying

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had the highest rate of non-peroxidized viable spermatozoa (Bodipy- / PI-), whereas vitrification,

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followed by freezing resulted in larger populations of peroxidized non-viable spermatozoa (Bodipy+ /

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PI+). In this regard, LPO has been attributed to equine sperm freezing due to factors such as increased 8

production of reactive species of oxygen and the activation of processes similar to apoptosis [31].

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However, in the present study vitrification had a more severe effect on sperm LPO than freezing.

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Additionally, freeze-drying caused lower levels of LPO associated with a population of viable

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spermatozoa when compared with vitrification and freezing. This result is paradoxical, considering the

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loss of motility observed in freeze-dried spermatozoa. Nevertheless, it is known that freeze-drying

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may cause fusion and loss of saturated and unsaturated fatty acids in biological membranes [56, 57],

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which could explain the lower presence of peroxidized lipids. The alteration in the integrity of the

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plasma membrane of the sperm could be related to the loss of cytosolic sperm factors involved in

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oocyte activation, such as phospholipase C-zeta (PLCζ) [18], which induces the oscillations of calcium

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in the oocyte and therefore triggers its activation [58]. This could explain the low rates of embryo

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production through ICSI with lyophilized sperm, consequently, the use of cytosolic sperm extract and

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chemical activators such as ionomycin has been evaluated to improve the production of blastocysts

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[18, 59].

Table 3.

Lipid peroxidation of frozen, vitrified and freeze-dried equine sperm.

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Treatment

Bodipy- / PI- (%)

Bodipy- / PI+ (%)

Bodipy+ / PI- (%)

Bodipy+ / PI+ (%)

Freezing

10.34 ± 2.69b

19.09 ± 10.18a

21.92 ± 13.09a

47.93 ± 13.42b

Vitrification

7.07 ± 2.00b

13.27 ± 9.41a

3.17 ± 0.93a

76.09 ± 10.01a

Freeze-drying

35.98 ± 7.01a

6.64 ± 2.44a

20.32 ± 6.00a

34.82 ± 9.41b

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N,N-Dimethylformamide. Vitrification: direct immersion in liquid nitrogen in an extender

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supplemented with 5% of bovine serum albumin and 0.25 M of sucrose. Freeze-drying: 48 h freeze-

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drying cycle (condensation -50 °C, vacuum pressure 150 mTorr) in a medium composed of DMEM/F-

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12 and 10% of bovine fetal serum. Bodipy- / PI-: non-peroxidized, viable spermatozoa. Bodipy- / PI+:

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non-peroxidized, non-viable spermatozoa. Bodipy+ / PI-: peroxidized, viable spermatozoa. Bodipy+ /

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PI+: peroxidized, non-viable spermatozoa. Results are shown as mean ± standard error of the mean.

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Different letters indicate statistically significant differences (p <0.05).

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It has been reported that freezing, vitrification and freeze-drying produce severe alterations in the cell

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membrane [13, 40, 57]. Likewise, the three methods employed in this study generated a high

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population of spermatozoa with an altered cell membrane and, consequently, a smaller population of

29

viable spermatozoa. Figures 1 and 2 show a representative analysis of the spermatozoa population

30

distribution by viability based on the assessment of their ∆ΨM and LPO.

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Figure 1. Representative analysis by flow cytometry of the viability of frozen, vitrified and freeze-

3

dried spermatozoa in relation to their mitochondrial membrane potential (∆ΨM). Freezing:

4

programmable freezing in a extender supplemented with 5% centrifuged egg yolk and 5% N,N-

5

Dimethylformamide. Vitrification: direct immersion in liquid nitrogen in an extender supplemented

6

with 5% of bovine serum albumin and 0.25 M of sucrose. Freeze-drying: 48 h freeze-drying cycle

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(condensation -50 °C, vacuum pressure 150 mTorr) in a medium composed of DMEM/F-12 and 10%

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of bovine fetal serum. Membrane Damage: non-viable spermatozoa with no ∆ΨM. DIOC6low:

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spermatozoa with less viability and low ∆ΨM. Viable: Population of viable spermatozoa with medium

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and high-∆ΨM.

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Figure 2. Representative analysis by flow cytometry of the viability of frozen, vitrified and freeze-

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dried spermatozoa in relation to their degree of lipid peroxidation. Freezing: programmable freezing in

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a extender supplemented with 5% centrifuged egg yolk and 5% N,N-Dimethylformamide.

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Vitrification: direct immersion in liquid nitrogen in an extender supplemented with 5% of bovine

17

serum albumin and 0.25 M of sucrose. Freeze-drying: 48 h freeze-drying cycle (condensation -50 °C,

18

vacuum pressure 150 mTorr) in a medium composed of DMEM/F-12 and 10% of bovine fetal serum.

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Dead: population of non-viable spermatozoa with lower or higher degree of lipid peroxidation. Viable:

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population of viable spermatozoa with lower or higher degree of lipid peroxidation.

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It is well documented that there is a negative correlation between defective sperm chromatin structure

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(DNA breaks) and fertility [60]. In this research, the DNA integrity of the spermatozoa was affected 10

ACCEPTED MANUSCRIPT by the preservation method employed (p <0.05) (see Table 4). Vitrified sperm had a higher rate of

2

spermatozoa with fragmented DNA when compared with frozen and freeze-dried sperm. However, the

3

DNA fragmentation index (DFI) was generally low for all preservation methods in comparison with

4

other studies conducted on frozen [51], freeze-dried [18] and vitrified [16] equine sperm.

5

Nevertheless, this could be attributed to the ability that the various methodologies had to detect single

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and double strand DNA fragmentation, since the technique employed in the present study detects

7

spermatozoa with the latter. On the other hand, low DFI levels have been associated with low

8

susceptibility to chromatin denaturation and, therefore, high sperm quality [61]. Additionally, no

9

differences were observed in the DNA damage level of the frozen, vitrified and freeze-dried

10

spermatozoa, which was expressed in the cytometric analysis as mean fluorescence intensity (MFI).

11

Figure 3 shows a representative analysis of the DNA fragmentation observed in the frozen, vitrified

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and freeze-dried equine spermatozoa.

13 Table 4.

Assessment of the DNA integrity of equine sperm.

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Treatment Freezing Vitrification Freeze-drying

DFI (%)

MFI

0.03 ± 0.01b

25250.00 ± 3287.23a

0.12 ± 0.04a

24462.50 ± 2500.70a

0.02 ± 0.01b

26500.00 ± 3262.41a

Freezing: programmable freezing in a extender supplemented with 5% centrifuged egg yolk and 5%

17

N,N-Dimethylformamide. Vitrification: direct immersion in liquid nitrogen in an extender

18

supplemented with 5% of bovine serum albumin and 0.25 M of sucrose. Freeze-drying: 48 h freeze-

19

drying cycle (condensation -50 °C, vacuum pressure 150 mTorr) in a medium composed of DMEM/F-

20

12 and 10% of bovine fetal serum. DFI: DNA fragmentation index. MFI: mean fluorescence intensity.

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Results are shown as mean ± standard error of the mean. Different letters indicate statistically

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significant differences (p <0.05).

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Figure 3. Representative analysis by flow cytometry of the DNA fragmentation of frozen, vitrified and

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freeze-dried spermatozoa. Freezing: programmable freezing in a extender supplemented with 5% 11

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centrifuged egg yolk and 5% N,N-Dimethylformamide. Vitrification: direct immersion in liquid

2

nitrogen in an extender supplemented with 5% of bovine serum albumin and 0.25 M of sucrose.

3

Freeze-drying: 48 h freeze-drying cycle (condensation -50 °C, vacuum pressure 150 mTorr) in a

4

medium composed of DMEM/F-12 and 10% of bovine fetal serum. 2n: somatic cells.
5

(Fragmented): spermatozoa with fragmented DNA. n: non-fragmented DNA.

6 Although DNA damage has been attributed to factors such as thermal shock, ice crystal formation and

8

oxidative stress, it has been described that vitrification of human and equine sperm does not cause

9

significant alterations of their integrity [16, 62]. It is known that the fragmentation of DNA in freeze-

10

dried sperm is one of the causes of decreased in vitro developmental ability of injected oocytes to the

11

blastocyst stage [63]; however, it has been reported that equine sperm appears to be resistant to DNA

12

fragmentation resulting from freeze-drying compared to conventional freezing and thawing

13

procedures, and even centrifugation [18]. This is consistent with the findings of the present study,

14

where lower DFI values were observed for freeze-dried sperm. It has been suggested that the damage

15

in the DNA of the freeze-dried sperm may be induced by the action of endonucleases released during

16

such process or by the oxidative stress occurring after rehydration [64]. As an alternative, the sperm

17

freeze-drying medium has been supplemented with calcium chelating agents such as 2,2′,2″,2‴-

18

(Ethane-1,2-diyldinitrilo) tetraacetic acid (EDTA) and EGTA, which act as inhibitors of endonuclease

19

activity [63]. A study on equine sperm freeze-drying found that EGTA provides a higher protective

20

effect for DNA than EDTA [26].

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The principal component analysis (which included two components) explained 64.5% of data

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variability. This analysis considered nine variables related to the DFI, ∆ΨM and LPO of the sperm

24

samples subjected to the three preservation methods (see Figure 4). This analysis showed that, in most

25

cases, vitrification simultaneously produced non-viable spermatozoa without mitochondrial activity

26

(PI+), peroxidized and non-viable spermatozoa (Bodipy+ / PI+) and spermatozoa with fragmented

27

DNA (DFI) (see Figure 4). In addition, samples of frozen, vitrified, and freeze-dried sperm showed

28

alterations that were either less severe and simultaneous or non-simultaneous. Moreover, some of

29

these samples simultaneously had low mitochondrial activity (low-∆ΨM) and non-peroxidized cells

30

(Bodipy- / PI+). Furthermore, freeze-dried sperm samples had the highest viability due to the

31

preservation of their mitochondrial activity (high-∆ΨM and medium-∆ΨM) without lipid peroxidation

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(Bodipy- / PI-). Based on these facts, the individual effect of the equine could have been associated

33

with the tolerance of the sperm to each preservation method, as stated in previous reports [11].

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Figure 4. Principal components between mitochondrial membrane potential, lipid peroxidation and

3

DNA fragmentation of equine spermatozoa. F: frozen spermatozoa. V: vitrified spermatozoa. L:

4

freeze-dried spermatozoa. High-∆ΨM: spermatozoa with high mitochondrial membrane potential.

5

Medium-∆ΨM: spermatozoa with medium mitochondrial membrane potential. Low-∆ΨM:

6

spermatozoa with low mitochondrial membrane potential. Bodipy- / PI-: non-peroxidized, viable

7

spermatozoa. Bodipy- / PI+: non-peroxidized, non-viable spermatozoa. Bodipy+ / IP-: peroxidized,

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viable spermatozoa. Bodipy+ / PI+: peroxidized, non-viable spermatozoa. PI+: non-viable

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spermatozoa with no mitochondrial activity. DFI: DNA fragmentation index.

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According to the results of the present study, freezing may be considered the preservation method that

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best protects equine sperm integrity and motility at the same time, in spite of the decrease in

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mitochondrial activity and the increase in LPO. Vitrification, in turn, may be considered the least

14

effective preservation method, since it produces more alterations in the mitochondrial activity and

15

DNA integrity of equine spermatozoa when compared with freezing and freeze-drying. Furthermore, it

16

also causes a dramatic decrease in sperm motility and kinetics. On the other hand, in spite of the loss

17

of motility, freeze-drying has a very good ability to preserve the mitochondrial activity and DNA

18

integrity of the sperm, as well as to reduce the peroxidation of the lipids in the cell membrane. Thus,

19

freeze-drying may be considered a promising method for long-term preservation of equine sperm,

20

which is an addition to its list of benefits, namely: advantages in terms of sample transportation,

21

worker safety and reduced cost [25]. However, further studies are needed to evaluate the causes of low

22

blastocyst production through ICSI with lyophilized equine spermatozoa. The present study is the first

23

report assessing the ∆ΨM and LPO of freeze-dried and vitrified equine spermatozoa.

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4. Conclusions

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When compared with freezing and vitrification, freeze-drying generates less lipid peroxidation and

2

leads to higher preservation of the mitochondrial membrane potential of equine sperm.

3 Conflict of interest

5

The authors have no conflict of interest to declare.

6

Acknowledgments

7

The authors would like to thank Politécnico Colombiano Jaime Isaza Cadavid for its financial support

8

and the Flow Cytometry Laboratory of the University of Antioquia for its technical support.

9

References

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[63] Nakai M, Kashiwazaki N, Takizawa A, Maedomari N, Ozawa M, Noguchi J, et al. Effects of chelating agents during freeze drying of boar spermatozoa on DNA fragmentation and on developmental ability in vitro and in vivo after intracytoplasmic sperm head injection. Zygote 2007;15:15-24.

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Highlights

Freeze-drying produces low lipid peroxidation in equine sperm. Freeze-drying can preserve the mitochondrial membrane potential of equine sperm.

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Freezing is highly effective for preserving equine sperm integrity.

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Vitrification causes severe damage to equine sperm.

ACCEPTED MANUSCRIPT Animal Welfare/Ethical statement: All procedures were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, or the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications

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No. 8023, revised 1978). Giovanni Restrepo Elizabeth Varela

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Juan Duque

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Mauricio Rojas