Oxidative stress at different stages of two-step semen cryopreservation procedures in dogs

Accepted Manuscript Oxidative stress at different stages of two-step semen cryopreservation procedures in dogs C.F. Lucio, F.M. Regazzi, L.C.G. Silva, D.S.R. Angrimani, M. Nichi, C.I. Vannucchi PII:

S0093-691X(16)00029-7

DOI:

10.1016/j.theriogenology.2016.01.016

Reference:

THE 13484

To appear in:

Theriogenology

Received Date: 15 June 2015 Revised Date:

11 January 2016

Accepted Date: 12 January 2016

Please cite this article as: Lucio CF, Regazzi FM, Silva LCG, Angrimani DSR, Nichi M, Vannucchi CI, Oxidative stress at different stages of two-step semen cryopreservation procedures in dogs, Theriogenology (2016), doi: 10.1016/j.theriogenology.2016.01.016. 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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Oxidative stress at different stages of two-step semen cryopreservation procedures in dogs

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C.F. Lucio a, F.M. Regazzi a, L.C.G. Silva a, D.S.R. Angrimani a, M. Nichi a, C.I. Vannucchi a,*

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São Paulo, Rua Prof. Orlando Marques de Paiva, 87 – Cidade Universitária, 05508-270, São Paulo,

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Brazil.

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* Corresponding author. Tel: +55 11 30911423; Fax: +55 11 30911437. E-mail address: [email protected] (C.I. Vannucchi)

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Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of

Abstract

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Sperm cryopreservation generates sperm damage and reduced fertilization capacity as consequence of

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reactive oxygen species formation. Identifying the critical points of the process will contribute to the

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development of strategies for oxidative stress prevention. Therefore, the aim of this experiment was to

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verify the occurrence of oxidative stress during the two-step cryopreservation process in dogs. Six healthy

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mature dogs were used and submitted to the two-step sperm cryopreservation protocol. The sperm

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analysis was done at three time points: after refrigeration, after glycerolization and after thawing by

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sperm motility (CASA), measurement of spontaneous and induced oxidative stress, sperm mitochondrial

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activity, plasma membrane integrity, flow cytometric evaluation of plasma and acrosome membrane

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integrity, mitochondrial membrane potential and sperm chromatin structure assay. There was an increase

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in free radical production after glycerolization (87.4 ±15.5 ng/mL of spontaneous TBARS after

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refrigeration and 1226.3 ±256.0 ng/mL after glycerolization; P<0.05), in association with loss of sperm

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mitochondrial activity. However, frozen-thawed samples had lower sperm motility, lower resistance to

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oxidative stress (448.7 ±23.6 ng/mL of induced TBARS after glycerolization and 609.4 ±35.9 ng/mL

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after thawing; P<0.05) and increased lipid peroxidation (4815.2 ±335.4 ng/mL of spontaneous TBARS

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after thawing; P<0.05) as well as increased damage to plasma and acrosomal membranes, compared to

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refrigeration and glycerolization. In conclusion, the production of free radicals by sperm cells begins

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during glycerolization. However, sperm oxidative damage intensifies after thawing. Despite intracellular

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ice formation during cryopreservation, the increased production of reactive oxygen species can be the

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explanation of the decrease in sperm motility, reduced mitochondrial activity and sperm plasma

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membrane and acrosomal damage.

33 Keywords: oxidative stress; glycerolization; thawing; spermatozoa; cryopreservation; canine

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1.

Introduction

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Sperm cryopreservation can induce the formation of free radicals mainly due to heat shock,

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exposure to atmospheric oxygen and the removal of seminal plasma [1]. Such procedures lead to lipid

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peroxidation of sperm cells and increased formation of reactive oxygen species [2]. Spermatozoa are

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particularly sensitive to oxidative stress, as their plasma membrane contains large amounts of

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polyunsaturated fatty acids, and the cytoplasm has low concentrations of protective enzymes [8].

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Therefore, the physiological concentration of antioxidant in the spermatozoa cytoplasm is not sufficient to

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protect against the cryopreservation process [9].

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The excessive production of free radicals is responsible for changes in semen quality, such as

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destruction of the lipid matrix structure, leading to a loss of membrane integrity, decreased sperm

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motility, sperm DNA damage and, ultimately, cell apoptosis and reduced fertilization capacity [3-7]. In

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dogs, the major consequences of lipid peroxidation of frozen-thawed semen are increased

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phosphatidylserine translocation index, higher intracellular hydrogen peroxide level and DNA

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fragmentation compared to fresh semen [10]. However, it is not known which step of semen

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cryopreservation is more prone to oxidative stress and thus generates higher sperm damage in dogs.

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Seminal cryopreservation protocols entail distinct steps that enable sperm reduction of

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temperature, dehydration and intra- and extra-cellular medium freezing. For the cryopreservation of

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canine semen, there is not one standard protocol, but well stablished protocols have been developed over

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the years, with tris-egg yolk extender and 8 to 10% glycerol as cryoprotectant [14, 17, 18, 19]. When

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compared to other cryoprotectants (such as ethylene glycol or dimethylformamide), glycerol shows higher

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post-thaw motility and fertilization capacity in dogs [20-22]. However, regardless the cryoprotectant used,

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canine frozen-thawed sperm present low viability and motility; and high susceptibility to decreased

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temperature, osmotic stress, cryoprotectant toxicity and intra- and extra-cellular ice formation. For

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example, canine sperm motility decreases markedly within 1 h after thawing, while chilled sperm can

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maintain fertilization capacity for up to 3 days [11-13]. Therefore, freezing and thawing procedures lead

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to significant decrease in sperm motility, viability, normal morphology and membrane integrity in dogs

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[10]. However, data on fertility rate of frozen-thawed semen are scarce in dogs, which hinders an overall

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analysis of the success of sperm cryopreservation [14]. Few studies have reported the fertilization

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potential of frozen-thawed semen used for artificial insemination [14-16].

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Knowing that the sperm cryopreservation process generates inherent seminal damage,

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identifying the critical points of the process will contribute to the development of strategies for oxidative

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stress prevention by the additional use of antioxidants in the sperm freezing extender. Therefore, the aim

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of this experiment was to verify the occurrence of oxidative stress during the two-step cryopreservation

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process in dogs, seeking to identify the critical step for sperm functionality.

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2.

Materials and Methods

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2.1. Animals and Experimental Design

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The current study was approved by the Bioethics Committee of the School of Veterinary

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Medicine and Animal Science – University of São Paulo. Six dogs, aged from 2 to 7 years, of distinct breeds and body weights were used. All males were

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healthy at the time of the study, free of anatomic or reproductive disorders. Only dogs showing breeding

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soundness including normospermia (sperm concentration of 500 million sperm per ejaculate and sperm

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motility higher than 70%) and adequate sexual behavior were selected for the study, but no prior selection

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based on semen freezability was performed. Two semen samples from each dog were obtained with a

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weekly interval and each ejaculate was processed separately.

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To verify the influence of oxidative stress in each specific step of canine sperm cryopreservation,

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the experimental design consisted of sperm analysis at three time points: after refrigeration, after

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glycerolization and after thawing. Therefore, the two-step sperm cryopreservation protocol was adopted

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as follows: Step A - extender without a cryoprotectant [0.26M Tris-hydroxymethyl-aminomethane,

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0.14M Citric acid monohydrate, 0.06M D-fructose, 20% egg yolk and 0.02M of gentamicin] for sperm

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cooling to 5°C; Step B – Step A extender with 10% glycerol as a cryoprotectant.

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The sperm-rich fraction of the ejaculates was collected by digital manipulation directly into

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calibrated plastic tubes through plastic funnels. After sperm analysis, the ejaculate was centrifuged

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(200 xg, 5 minutes) and the supernatant discarded. The sperm pellet was resuspended in the extender

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without glycerol (Step A) at 37°C in a volume sufficient to achieve a sperm count of 200 million

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spermatozoa per mL. After dilution, the semen sample was slowly cooled from 37°C to 5°C (for

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approximately 1 h and 30 min). Sequentially, the extender with cryoprotectant (Step B) was added to the

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semen sample (also at 5°C) using the same previous volume employed for step A. Thus, the final glycerol

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concentration (5%) and total sperm concentration of 100 million spermatozoa per mL were reached. The

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sample was kept at 5°C for an additional 1 h for glycerolization to occur. The semen was then packaged

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in 0.5 mL straws, kept in nitrogen vapor (-70°C) for 20 min and sequentially immersed and stored in

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liquid nitrogen.

At no less than 15 days after cryopreservation, two straws from each semen sample were thawed in a water bath at 37°C for 30 sec in order to process with sperm analysis.

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2.2. Assessment of sperm motility

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Semen samples were evaluated for sperm motility by computer-assisted sperm analysis (CASA;

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HTM-IVOS-Ultimate 12.3; Hamilton Thorne Biosciences, Beverly, MA, USA), according to a previously

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described protocol [23]. Briefly, 10 µl of each sample was deposited into microscope slides previously

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warmed at 37 °C and covered by coverslips. Eight fields of view were randomly selected and evaluated

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for the following parameters, previously described by Rijsselaere et al. [24]: velocity average pathway

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(VAP; µm/s), curvilinear velocity (CSL/VCL; µm/s), velocity straight line (VSL; µm/s), amplitude of

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lateral head displacement (ALH; µ/M), beat cross-frequency (BCF; Hz), straightness (STR; %), linearity

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(LIN; %); total motility (MOT; %) and progressive motility (PROG; %). Sperm was additionally divided

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into 4 groups based on spermatozoa speed: fast (RAP%; VAP> 50 µm/s), medium (MED%, 30 µm/s

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2.3. Oxidative stress evaluation

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The measurement of thiobarbituric acid reactive substances (TBARS) aims to assess

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malondialdehyde (MDA) levels, a product of lipid peroxidation, called spontaneous TBARS. This was

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performed in accordance to the protocol first described by Ohkawa et al. [25]. The method measures

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indirectly the amount of sperm oxidative stress by analyzing the concentration of MDA; two molecules of

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thiobarbituric acid reacts with one molecule of malondialdehyde, at high temperatures and low pH,

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resulting in a pink chromogen that can be quantified with a spectrophotometer. To precipitate proteins,

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200 µl of pre-centrifuged supernatant seminal sample and 400 µl of a 10% solution (v:v) of trichloroacetic

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acid (TCA 10%) were mixed and centrifuged (18000 g for 15 min at 15°C). After centrifugation, 500 µl

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of the supernatant and 500 µl of 1% (v:v) thiobarbituric acid (TBA, 1%) in 0.05N sodium hydroxide were

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placed in glass tubes, boiled in a water bath (100°C) for 10 min, and subsequently cooled in an ice bath

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(0°C) to stop the chemical reaction. The TBARS were then quantified using a spectrophotometer at a

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wavelength of 532 nm. The results were compared to a standard curve previously prepared with a

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standard solution of malondialdehyde (MDA). The TBARS concentration was determined using the value

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of 1.56 × 105/M/cm as the MDA extinction coefficient [26]. The lipid-peroxidation index was described

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as nanograms of TBARS/mL.

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To analyze the susceptibility of sperm to oxidative stress and indirect antioxidant capacity (so

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called induced TBARS), we followed the protocol described by Nichi et al. [27], in which sperm were

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submitted to a challenge with a ROS generating system. In brief, 0.5 mL of the previously prepared sperm

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suspension containing 1x106/mL of TALP was incubated with ferrous sulfate (125 µL, 4 mM) and

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sodium ascorbate (125 µL, 20 mM) for 1.5 hours at 37°C. Subsequently, 10% TCA at 4°C was added,

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and the mixture was centrifuged (18,000 xg, 15 min) to promote the precipitation of proteins. Analysis

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was performed by mixing 500 µL of the previously prepared supernatant with 500 µL of 1%

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thiobarbituric acid (TBA 1% diluted in 0.05 M sodium hydroxide) in a water bath kept at 90-100°C for 15

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min and then immediately cooled in an ice bath (0oC) to interrupt the chemical reaction. The

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thiobarbituric acid reactive substances (TBARS) were quantified spectrophotometrically at a wavelength

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of 532 nm (Ultrospec 3300 Pro ®, Amersham Biosciences), as previously described. The lipid-

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peroxidation index was described as nanograms of TBARS/106sperm.

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2.4. Evaluation of mitochondrial activity and plasma membrane integrity

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Mitochondrial activity was analyzed using 3.3′diaminobenzidine (DAB) staining [28]. An

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aliquot of each sample was incubated with a DAB solution (1 mg/mL of DAB in PBS) in an amber

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microcentrifuge tube (1:1) for 1 h at 37°C. After incubation, the mixture was smeared onto microscopy

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slides and fixed in 10% formalin for 10 min. Slides were then examined under a phase contrast

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microscope (x1000). A total of 200 sperm cells were counted and classified in four different classes:

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Class I with 100% of the mid-piece stained indicating full mitochondrial activity; Class II with more than

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50% of the mid-piece stained indicating medium activity; Class III with less than 50% of the mid-piece

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stained indicating low activity; and Class IV with the absence of staining in the mid-piece indicating no

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mitochondrial activity.

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Samples were also evaluated for plasma membrane integrity using the eosin/nigrosin stain [29], which revealed the percentage of live/dead spermatozoa by counting 200 sperm cells (x1000).

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2.5. Assessment of sperm membrane integrity, mitochondrial membrane potential and sperm

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chromatin structure assay

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Flow cytometry was performed with the use of the Guava EasyCyteTM Mini System (Guava®

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Technologies, Hayward, CA, USA) with fluorescent probes to specifically analyze each sperm parameter,

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with a 488 nm laser argon and the following filters (photodetector): PM1 (583 nm) to yellow

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fluorescence, PM2 (680 nm) to red and PM3 (525 nm) to green. For data analysis, cell doublets and

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debris were excluded using PM3/FSC (forward scatter). A minimum of 20,000 spermatozoa were

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examined for each assay. All of the data were analyzed using FlowJo v8.7 Software (Flow Cytometry

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Analysis Software – Tree Star Inc., Ashland, Oregon, USA). In order to validate flow cytometry protocols

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for the assessment of sperm membrane integrity and mitochondrial membrane potential, we followed the

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procedures described by Celeghini et al [30], modified for dogs. In brief, the positive control was

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obtained by submitting fresh canine semen to 10 cycles of freezing and thawing in liquid nitrogen in order

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to provoke disruption of membranes; whereas the negative control was non-processed fresh canine sperm

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samples.

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Plasma and acrosome membrane integrity were assessed using the probes propidium iodide (PI-

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Sigma Chemical Co., St. Louis, MO) and Pisum sativum agglutinin conjugated with fluorescein

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isothiocyanate (FITC-PSA-Sigma Chemical Co., St. Louis, MO), according to the technique adapted by

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Celeghini et al. [30]. Seven million sperm cells were resuspended in 150 µL of TALPm [Modified

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Tyrode’s Albumin Lactate Pyruvate (0.1 M NaCl, 0.003 M KCl, 0.0004 M MgCl2, 0.0003 M NaH2PO4,

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0.025 M NaHCO3, 0.003 M CaCl2H2O, 0.3% v/v lactic acid SYR, 0.01 M Hepes, pH 7.4)] with 50 µL of

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FITC-PSA (100 µg/mL in Dulbecco’s phosphate-buffered saline solution - DPBS – with 10% sodium

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azide solution) and 2 µL of PI (0.5 mg/mL in DPBS). After 8 min at 37°C, 300 µL of TALP was added to

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the solution, and it was analyzed by flow cytometry. The sperm population was selected on a FITC-PSA

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probe graph, and we subsequently measured points on the graph with red staining (PI) and green (FITC-

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PSA). Populations were classified as unmarked by the probe, labeled only with FITC-PSA, corresponding

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to the green color, marked only by PI probe, having a red color, and double-labeled (FITC-PSA + PI).

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Acrosome damage was characterized by a green color and plasma membrane damage by a red color.

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The mitochondrial membrane potential was determined with the use of 5, 5’, 6, 6’, tetrachloro-1,

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1’, 3, 3’–tetraethyl-benzimidazolcarbocyanine iodide (JC-1; SigmaAldrich Chemical Co., Germany) [30].

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For this purpose, 2 µL of JC1 probe (1 µM in DMSO) was added to 7 million spermatozoa. After 8 min at

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37°C, 300 µL of TALP was added and analyzed by flow cytometry. The sperm population was selected

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by red fluorescence and then evaluated in the dot plot for yellow fluorescence. Sperm were separated into

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two populations: high mitochondrial membrane potential and low mitochondrial membrane potential. In

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spermatozoa with high mitochondrial potential, JC-1 formed complexes known as J-aggregates, with

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intense red fluorescence. For sperm with low mitochondrial membrane potential, JC-1 remained in the

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monomer form, which appears only as green fluorescence.

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Sperm chromatin susceptibility to acid-induced denaturation was assessed based on the sperm

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chromatin structure assay (SCSA) [31]. Chromatin instability was quantified by flow cytometric

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measurement of the metachromatic shift from green (double-strand DNA) to red (denatured single-strand

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DNA) of acridine orange (AO) fluorescence. The samples were diluted with 100  µL TNE buffer (0.01  M

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Tris–HCl, 0.15  M NaCl, 1  mM EDTA, pH 7.4) and mixed with 400  µL of an acidified detergent solution

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(0.08  M HCl, 0.1% Triton X-100, 0.15  M NaCl, pH 1.2). After 30 sec, spermatozoa was stained by

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adding 600  µL of an AO staining solution (0.037  M citric acid, 0.126  M Na2HPO4, 0.0011  M disodium

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EDTA, 0.15  M NaCl, pH 6.0). After 5 min of staining, the samples were examined by flow cytometry.

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2.6. Statistical analysis

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All data were evaluated using SAS System for Windows 9.3 (SAS Institute Inc., Cary, NC,

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USA). The effect of moment of evaluation (post-refrigeration, post-glicerolization and post-thaw) was

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determined using parametric and non-parametric tests, according to the residue normality (Gaussian

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distribution) and variance homogeneity of each variable. Data was transformed whenever necessary. A

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probability value of P < 0.05 was considered statistically significant. Spearman correlation test was used

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to calculate relationship between variables studied in each moment of evaluation. Results are reported as

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untransformed means ± S.E.M.

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Results

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The sperm thawing process negatively affected the sperm velocity average pathway and velocity

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straight line (VAP and VSL) as well as the percentage of sperm with progressive movement (PROGR)

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and fast velocity (RAPID; Table 1). On the other hand, glycerolization had a negative impact on velocity

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curvilinear (VCL), amplitude of lateral sperm head displacement (ALH), beat-cross frequency (BCF),

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percentage of motile sperm (MOT) and average velocity (MED) when compared to refrigerated samples

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(Table 1). There was a significant and progressive increase in the percentage of static spermatozoa

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(STAT) within cryopreservation process (Table 1).

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Lipid peroxidation, assessed by the TBARS assay on the basis of MDA formation, increased

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during glycerolization, which in turn was higher in post-thaw samples (Table 1). Therefore, the

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glycerolization step promotes more oxidative stress than refrigeration. Sperm resistance to oxidative

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stress, determined by the induced TBARS technique, reduced significantly in post-thaw sperm, indicating

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a decrease on antioxidant capacity (Table 1).

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Regarding the integrity of the sperm membrane, there was a lower percentage of sperm with an

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intact plasma membrane (eosin-nigrosine stain) and a higher percentage of plasma and acrosomal

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membrane injury (probes FITC / PI) after thawing (Table 1). No influence of glycerolization was

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observed in comparison to refrigeration (Table 1).

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In addition to the effect on membrane integrity, frozen-thawed sperm presented with decreased

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mitochondrial activity and mitochondrial membrane potential compared to refrigeration and

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glycerolization (Fig. 1). However, the addition of glycerol reduced the percentage of sperm cells with

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high and medium mitochondrial activity (DAB I and II) compared to refrigerated samples (Fig. 1).

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For the correlation analysis, we found a positive correlation between the percentage of static

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sperm and high mitochondrial membrane potential during cooling (r = 0.35, p = 0.04), plasma membrane

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damage (FITC / PI PM, r = 0.36, p = 0.03) and beat cross (BCF; r = 0.64, p <0.0001). Only in frozen-

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thawed sperm did we observe a positive correlation among the percentage of damaged plasma

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membranes (FITC / PI PM) or acrosomes (FITC / PI AM) and fast sperm velocity (r = 0.38, p = 0.02 and

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r = 0, 41, p = 0.01, respectively) and total motility (r = 0.43, p = 0.01 and r = 0.45, p = 0.0008,

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respectively). In addition, there was a positive correlation between the percentage of static spermatozoa

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and oxidative stress (spontaneous TBARS; r = 0.37, p = 0.03) and damaged plasma membrane and

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acrosome (FITC / PI PAM; r = 0.35, p = 0.04) after thawing.

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Discussion

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Several researches have reported the damage to sperm cells submitted to cryopreservation.

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However, no studies have determined the specific causes of sperm alteration in dogs, as well as the timing

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of when they occur. The generation of reactive oxygen species is identified as one of the main causes of

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sperm damage during cryopreservation [32]. Although exposure to liquid nitrogen and subsequent sperm

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thawing are considered critical moments for oxidative stress by several authors, we showed, for the first

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time, that the production of ROS emerges during glycerolization.

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The significant increase in free radical production after the equilibration period with the

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cryoprotectant, in association with loss of sperm mitochondrial activity, denote the negative influence of

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glycerol exposure. The use of intracellular cryoprotectant agents promotes imbalance in sperm

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bioenergetic metabolism, through ion pump alteration, interfering with the exchange of ions and the

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electric capacity of the sperm membrane and, ultimately, disrupting ATP production [33, 34].

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Furthermore, glycerolization subjects sperm to osmotic stress, which leads to changes in cell volume,

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compromising mainly sperm motility [35, 36]. Therefore, the osmotic stress associated with sperm

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bioenergetic changes due to glycerol addition caused a reduction in mitochondrial activity and increased

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the formation of free radicals, leading to membrane lipid peroxidation and a consequent loss of sperm

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motility and velocity. In fact, in the present work, glycerolization reduced the percentage of motile sperm

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and speed, and increased the number of static spermatozoa.

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Regardless of the fact that cryopreservation reduces sperm defense capacity against oxidative

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stress by removing seminal plasma antioxidants [32, 37], Neagu et al [38], suggested an individual effect

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in the canine sperm susceptibility to the oxidative insult during cryopreservation. In this experiment, it is

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interesting to note that no changes in the susceptibility of sperm cells to oxidative stress occurred after

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glycerolization. This finding allows us to suggest the maintenance of the antioxidant defense mechanisms

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of canine sperm during the stabilization with cryoprotectants. It is possible to infer, therefore, that canine

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sperm has transient cytoplasmic antioxidant reserves, which are consumed during glycerolization.

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However, further experiments comparing the removal or maintenance of seminal plasma on sperm

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susceptibility to free radicals, as well as the addition of antioxidants during the cryopreservation process,

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are needed to test this hypothesis.

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We observed increased sperm damage after thawing as evidenced by an increase in the

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percentage of sperm showing low or absent mitochondrial activity. Moreover, frozen-thawed sperm had a

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decreased mitochondrial membrane potential, showing an overall compromised mitochondrial function in

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comparison to refrigeration and glycerolization. In addition to the oxidative and metabolic changes

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previously described during glycerolization, a modification of the sperm aqueous fraction from the liquid

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to solid-state occurs during freezing. This physical alteration leads to an increase in solute concentration

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of the aqueous medium and, consequently, an accentuated osmotic stress, promoting changes in cellular

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metabolism [39]. We believe that these changes negatively influence energetic production of sperm cells

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because we observed a significant reduction in sperm mitochondrial membrane potential after

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cryopreservation. Mitochondrial membrane potential is maintained by the transfer of electrons along the

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transport chain, which promotes the extrusion of protons across the mitochondrial membrane. The energy

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produced by the difference in protons concentration, called proton motive force, is used for ATP

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formation [40]. Therefore, we verified that frozen-thawed sperm have no ability to maintain the

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electrochemical proton gradient across mitochondrial membranes, and thus are unable to adequately

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produce ATP.

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Although sperm oxidative stress was initiated after the addition of glycerol, frozen-thawed

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samples were more intensely affected. After thawing, there was a significant increase in the percentage of

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sperm with low mitochondrial activity. Sperm with damaged mitochondria (reduced mitochondrial

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activity – DAB class III) are great sources of free radicals [41, 42], which allows us to suggest a cause-

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consequence relationship. Thus, we believe that the increase in oxidative stress after thawing is due to

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increased generation of ROS by sperm with mitochondrial damage. Simultaneously with the energy generation deficit after thawing, the intense production of free

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radicals results in reduction of sperm motility. In fact, we observed a decrease in the velocity average

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pathway (VAP), velocity straight line (VSL), velocity curvilinear (VCL) and amplitude lateral head

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(ALH) of frozen-thawed sperm. Sperm motility is one of the most important parameters in sperm

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fertilizing capacity, expressing their viability and structural integrity [23]. Therefore, our results suggest a

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reduction in the fertilizing capacity of frozen-thawed canine sperm. Additionally, positive correlation was

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found between spontaneous oxidative stress and the percentage of static sperm after thawing. Thus, the

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cryopreservation process results in the appearance of a greater amount of dead and defective sperm,

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capable of generating reactive oxygen species [43], which therefore becomes a vicious cycle.

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In our study, frozen-thawed samples had lower sperm motility, lower resistance to oxidative

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stress and increased lipid peroxidation as well as increased damage to plasma and acrosomal membranes,

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compared to refrigeration and glycerolization. According to Bansal and Bilaspuri [9], excessive oxidative

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stress directly alters the plasma membrane integrity of spermatozoa by promoting lipid peroxidation and

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consequently impairing sperm function. The intense production of free radicals, cold shock and the

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formation of intracellular ice crystals during cryopreservation may also be causes of the plasma

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membrane injury [44].

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Changes in cellular permeability during different stages of sperm cryopreservation may favor

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calcium influx, simulating characteristics similar to those observed during sperm capacitation [45]. In

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fact, we observed a positive correlation between motility and progressive sperm movement, and sperm

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velocity with plasma membrane or acrosomal injury in frozen-thawed sperm. Thus, these data suggest a

319

pre-capacitation state of frozen-thawed sperm after cryopreservation. Moreover, there was a positive

320

correlation among the percentage of static spermatozoa, plasma membrane damage, high mitochondrial

321

potential (JC1) and beat-cross frequency (BCF) during refrigeration. These results taken together suggest

322

also a mechanical capacitation state with consequent hypermotility while cooling, characteristic of the

323

sperm capacitation process. Conversely, the percentage of static spermatozoa correlated positively after

324

thawing with oxidative stress and plasma and acrosomal membrane damage. Thus, we can infer that

325

sperm are in the final stage of cell death or apoptosis at this time point, indicating a decrease in sperm

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resistance and changes in its microenvironment after cryopreservation. Hence, it is of utmost importance

327

to use frozen-thawed semen immediately for artificial insemination. Surprisingly, our results show fewer changes in the sperm chromatin after thawing compared to

329

those associated with glycerolization. Our results are in agreement with Koderle et al. [46], who detected

330

lower sperm DNA fragmentation after thawing compared to fresh canine semen. Additionally, Urbano et

331

al. [47] showed that DNA damage is not a direct effect of sperm cryopreservation in dogs. We believe

332

that the osmotic stress caused by glycerol resulted in changes in sperm chromatin conformation,

333

sensitizing the DNA strand to chemical damage. Therefore, while performing the acid challenge step of

334

the SCSA technique during glycerolization, chromatin damage increased and led to a greater binding

335

capacity of the SCSA probe. However, after thawing, there was a conformational reorganization of

336

chromatin, preventing the negative action of the acid challenge and the excessive binding of the dye to

337

sperm DNA. These results suggest a particular fragility of canine sperm to the acid challenge. Therefore,

338

we suggest careful use of the SCSA technique to assess the integrity of sperm chromatin in dogs.

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Taking the results together, we verified that sperm refrigeration in dogs does not promote

340

excessive oxidative stress, which suggests that the addition of antioxidants to seminal extender at this

341

time point or for such a seminal procedure is unnecessary. In fact, dog spermatozoa are considered

342

relatively resistant to cooling at 5oC and the action of free radicals in a balanced manner is considered

343

important for sperm hyperactivation and capacitation, and the acrosome reaction [48, 49]. Therefore,

344

antioxidant supplementation to this situation can generate oxidative imbalance, compromising the sperm

345

fertilizing capacity. On the other hand, the cryopreservation of canine sperm led to the formation of

346

excessive reactive oxygen species after the addition of glycerol, demonstrating the importance of

347

antioxidant supplementation to the extender medium simultaneously with inclusion of the cryoprotectant.

348

Several studies have evaluated the addition of antioxidants to semen cryopreservation medium with

349

positive results [50-52]. However, additional studies are needed in dogs to determine which type of ROS

350

is formed during sperm cryopreservation, making it is possible to define the concentration of the specific

351

antioxidants to control the ROS formation in canine semen.

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352 353 354

5.

Conclusion

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In conclusion, the production of free radicals by sperm cells begins during glycerolization.

356

However, sperm oxidative damage intensifies after thawing. Therefore, the increased production of

357

reactive oxygen species can be one of the main factors under sperm injuries observed during canine

358

semen cryopreservation, such as decreased sperm motility, mitochondrial activity and plasma membrane

359

and acrosomal damage.

360 Acknowledgments

362

This research was financially supported by FAPESP 2009/52760-3.

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ability of frozen-thawed bull spermatozoa. Int J Androl. 2008;31:40-9.

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[51] Gadea J, Molla M, Selles E, Marco MA, Garcia-Vazquez FA, Gardon JC. Reduced glutathione

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content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione

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to the freezing and thawing extenders. Cryobiology. 2011;62:40-6.

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[52] Perumal P, Selvaraju S, Selvakumar S, Barik AK, Mohanty DN, Das S, et al. Effect of pre-freeze

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addition of cysteine hydrochloride and reduced glutathione in semen of crossbred Jersey bulls on sperm

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parameters and conception rates. Reprod Domest Anim. 2011;46:636-41.

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482 483 Figure caption

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Fig. 1. Sperm mitochondrial activity analyzed by the oxidation of 3,3'-diaminobenzidine (DAB) and

486

sperm high mitochondrial potential (JC1 probe) after each step of the cryopreservation process

487

(refrigeration, glycerolization and thawing) in dogs (n = 6). Superscripts represent differences between

488

cryopreservation steps at P < 0.05.

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ACCEPTED MANUSCRIPT Table 1 Mean and standard error (X±SE) of the sperm computer analysis of motility (CASA), concentration of thiobarbituric acid reactive substances (spontaneous and induced TBARS), percentage of intact plasma membrane sperm (eosin-nigrosine stain – E/N), membrane integrity (FITC / PI probes) and sperm chromatin structure assay (SCSA) after each step of the cryopreservation process (refrigeration,

Refrigeration

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glycerolization and thawing) in dogs (n = 6).

Glycerolization

Thawing

98.4 ±4.5a

91.7 ±3.4a

67.8 ±2.5b

VSL (µm/s)

79.2 ±4.7a

78.7 ±3.8a

56.8 ±2.8b

VCL (µm/s)

160.8 ±5.1a

146.0 ±4.4b

114.7 ±3.5c

ALH (µm)

7.9 ±0.3a

7.1 ±0.3b

6.3 ±0.3b

BCF (Hz)

29.7 ±1.2ab

27.4 ±0.8b

31.9 ±0.5a

73.5 ±1. 9b

80.0 ±1.9a

76.6 ±1.7ab

68.9 ±3.3a

58.8 ±3.3b

20.7 ±2.0c

41.4 ±3.8a

43.1 ±3.2a

12.1 ±1.5b

54.3 ±4.0a

49.4±3.3a

13.8 ±1.6b

14.6 ±1.3a

9.2 ±1.2b

6.9±0.5b

18.8 ±2.5c

28.4 ±3.3b

60.4 ±3.7a

Spontaneous TBARS (ng/mL)

87.4 ±15.5c

1226.3 ±256.0b

4815.2 ±335.4a

Induced TBARS (ng/106sperm)

411.4 ±23.8b

448.7 ±23.6b

609.4 ±35.9a

Intact plasma membrane E/N (%)

65.7 ±3.6a

67.1 ±2.8a

40.9 ±4.9b

Intact plasma and acrosomal membrane (%)

33.3 ±2.5

36.9 ±2.1

34.6 ±2.4

Plasma membrane injury (%)

11.2 ±1.3a

11.1 ±1.1a

13.8 ±1.3b

Acrosomal membrane injury (%)

35.4 ±4.2a

32.1 ±2.8a

17.2 ±2.2b

Plasma and acrosomal membrane injury (%)

20.1 ±2.1b

20.0 ±1.9b

32.8 ±2.3a

SCSA – DNA fragmentation (%)

3.09 ±1.8ab

3.97 ±3.1a

2.69 ±1.7b

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RAPID (%) MEDIUM (%)

a,b,c

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VAP (µm/s)

Superscripts represent differences between cryopreservation steps at P < 0.05.

ACCEPTED MANUSCRIPT Refrigeration 80 70

Glycerolization

Thawing

a b

60 a a

50 c b

30 c

20

b

a

10

b

b

a

0 Low mitochondrial activity (DAB III)

Absent mitochondrial activity (DAB IV)

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Intermediate mitochondrial activity (DAB II)

b

SC

b High mitochondrial activity (DAB I)

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a

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High mitochondrial potential

ACCEPTED MANUSCRIPT

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This experiment has determined the specific causes of sperm alteration during cryopreservation in dogs, as well as the timing of when they occur. Glycerolization induces an increase in free radical production, in association with loss of sperm mitochondrial activity; The freezing step of cryopreservation lowers sperm resistance to oxidative stress and increas lipid peroxidation compared to refrigeration and glycerolization; The production of reactive oxygen species emerges during glycerolization of the cryopreservation protocol in dogs.

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