Effect of Caffeine and Pentoxifylline Added Before or After Cooling on Sperm Characteristics of Stallion Sperm

Journal Pre-proof Effect of caffeine and pentoxifylline added before or after cooling on sperm characteristics of stallion sperm Melissa Rossi, Raul Gonzalez-Castro, Maria Elena Falomo PII:

S0737-0806(19)30651-3

DOI:

https://doi.org/10.1016/j.jevs.2019.102902

Reference:

YJEVS 102902

To appear in:

Journal of Equine Veterinary Science

Received Date: 31 October 2019 Revised Date:

18 December 2019

Accepted Date: 20 December 2019

Please cite this article as: Rossi M, Gonzalez-Castro R, Falomo ME, Effect of caffeine and pentoxifylline added before or after cooling on sperm characteristics of stallion sperm, Journal of Equine Veterinary Science (2020), doi: https://doi.org/10.1016/j.jevs.2019.102902. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Inc.

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Effect of caffeine and pentoxifylline added before or after cooling on sperm

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characteristics of stallion sperm

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Melissa Rossia*, Raul Gonzalez-Castrob, Maria Elena Falomoc

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a

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Viale dell'Università 16, 35020 Legnaro (PD), Italy, e-mail: [email protected]

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b

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Fort Collins, Colorado 80521, USA, e-mail: [email protected]

Department of Animal Medicine, Production and Health (MAPS), Università di Padova,

Equine Reproduction Laboratory, Colorado State University, 3101 Rampart Road

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c

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Viale dell'Università 16, 35020 Legnaro (PD), Italy, e-mail: [email protected]

Department of Animal Medicine, Production and Health (MAPS), Università di Padova,

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(*) Corresponding author

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Melissa Rossi, DVM, PhD

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Viale dell'Università 16, Legnaro (PD), Italy,

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E-mail: [email protected]

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Tel: +39 049 8272608

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Abstract

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Different additives have been tested in cooled stallion sperm, in order to maintain

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sperm quality and to ameliorate the decrease in sperm fertility potential. In several species,

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caffeine and pentoxifylline promote sperm motility by increasing energy production. We

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evaluate the effects of caffeine and pentoxifylline when added to stallion sperm before or

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after cooling. Three ejaculates from five stallions each were processed and resuspended in

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skim milk extender. Caffeine (5 mM), pentoxifylline (3.5 mM) or both additives combined

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were included to sperm before or after cooling (4°C for 24 h). Cooled sperm were incubated

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at 37°C and evaluated at 0, 30, 60 and 120 min for motility, morphology, viability (flow

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cytometry) and membrane functionality (hypoosmotic swelling test). Results were analyzed

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by two-factor mixed model for repeated measures and Tukey comparisons. As main effects,

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the caffeine and pentoxifylline affected significantly motility and kinematic parameters,

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without interaction between treatment and incubation after cooling. No differences were

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observed whether the additives were added prior or after cooling. Pentoxifylline added after

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cooling reduced significantly motility during incubation, but with higher values at 30 min.

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We detected a decrease in morphologically normal sperm (P < 0.0001), caused by an

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increasing of tail defects (P < 0.003) in presence of both additives. Viability and membrane

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functionality were also significantly impaired by additives. Pentoxifylline when added after

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cooling, improved sperm motility and kinematic parameters for a short period of time.

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However, sperm characteristic related to fertility potential were compromised after a

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prolonged exposure to caffeine or pentoxifylline.

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Keywords: Caffeine, Motility, Pentoxifylline, Stallion, Viability.

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1. Introduction

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Artificial insemination of mares with cooled semen has changed the logistic of the

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equine breeding industry, becoming a routine practice. During shipping, stallion semen is

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extended and maintained at ~5°C for about 24 h. However, cooled sperm undergo a

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physiological decline in potential fertility, which generally is not retained longer than 24 to

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48 h [1]. Different additives such as ascorbic acid 2-glucoside, coenzyme Q, vitamin C and E,

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curcumin and many others have been tested, in order to maintain sperm characteristics

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associated to potential fertility in cooled stallion sperm [2–5].

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Caffeine (CF) and pentoxifylline (PT) are methylxanthine derivatives that when added

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to frozen-thawed stallion sperm has resulted in an improvement of total and progressive

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motility [6–8]. In stallion cooled sperm, PT increases total and progressive motility,

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progressive velocity, viability and plasma membrane integrity until 48 h, when compared to

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control group [9]. In boars, the inclusion of CF to the cooling extender for insemination

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improves both pregnancy and farrowing rates by transiently inhibiting migration of

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polymorphonuclear leukocytes into the uterine lumen [10,11].

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Caffeine and pentoxifylline inhibit cyclic nucleotide phosphodiesterase, resulting in an

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increase of cyclic adenosine monophosphate (cAMP) that promotes capacitation and

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spontaneous acrosome reaction and consequently increases sperm motility [7,12–14]. High

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cAMP level generates adenosine triphosphate (ATP) that is used as energy resource to sperm

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movement [7]. Additionally, CF may have a direct effect on cellular metabolism, and such

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effect depends on the concentration of calcium ions and the immediate hyperactivation of

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incubated sperm [15]. In sperm, cAMP activates protein kinase (PKA), resulting in protein

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tyrosine phosphorylation, which regulates and modulates pathways associated to capacitation

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[16].

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Pentoxifylline was used in sperm preparation for procedures in assisted reproduction in

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humans, resulting in increased motility and proportion of hyperactivated and acrosome

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reacted sperm, and also acting as antioxidant [17]. Pentoxifylline significantly improved

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fertilization rates and embryo formation in vitro when it was included to sperm preparations

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of fertile or infertile patients [18,19]. However, the extent of CF and PT on fertility in vivo

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remains to be elucidated.

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We hypothesized that the addition of CF and PT for cooled stallion sperm processing

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would result in improvement of sperm quality. The aim of this study was to evaluate the

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effects of CF, PT individually and both combined on sperm characteristics such motility,

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kinematic parameters, morphology, viability and membrane functionality when added to

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stallion sperm before or after a 24 h cooling period.

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2. Materials and methods

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2.1 Semen collection, processing and cooling

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Three ejaculates from five stallions each, between 3 and 25 years, were collected with

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an artificial vagina (model Missouri, Minitube, Germany). Immediately after collection, raw

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semen was filtered with a cellulose filter (Minitube) to remove the gel fraction. Volume and

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sperm concentration were assessed with a graduate cylinder and Spermacue Photometer

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(Minitube), respectively. Filtered semen was extender 1:1 in skim-milk extender (SM) [20]

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and centrifuged at 400g for 15 min. The supernatant was removed, and sperm pellet was

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resuspended in SM. Sperm obtained from each single ejaculate were split in control and

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treatments at a final concentration of 50 x106 sperm/mL. Sperm extended in SM without

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additives was considered as control. For treatments, resuspended sperm were supplemented

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with 5 mM caffeine (CF, Millipore Sigma, St. Louis, MO), 3.5 mM pentoxifylline (PT,

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Millipore Sigma) or 5 mM CF plus 3.5 mM PT (CFPT), added before or after 24 h cooling,

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according to previous studies [7,21–25]. Osmolarity of extenders used for control and

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treatments ranged between 301 and 315 mOsm/kg; and pH between 7.0 and 7.2. To avoid

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exposure to light, samples were wrapped in aluminum foil and then cooled down in a

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refrigerator to 4°C for 24 h. Temperature inside the refrigerator was recorded every 10 min by

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a temperature data logger (Elitech® RC-5, London, UK). During the cooling period, the

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refrigerator temperature ranged between 4.1 and 4.6°C, and samples were at 4.1°C at the end

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of the cooling period. After 24 h, cooled sperm samples containing CF, PT or CFPT were

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incubated for 15 min at 37°C in water bath before starting the assessment and were

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maintained at 37°C during 120 min incubation. Cooled samples for additive after cooling

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were incubated for 10 min at 37°C in a water bath, before CF, PT and CFPT were added to

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sperm and incubated for additional 15 min at 37°C before assessment. Samples were assessed

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at 0, 30, 60 and 120 min for motility, morphology, viability and HOS test.

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2.2 Sperm motility Percentage of sperm total motility, average path velocity (VAP, µm/s), curvilinear

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velocity (VCL, µm/s), straight-linear velocity (VSL, µm/s), amplitude of lateral head

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displacement (ALH, µm), straightness (STR, VSL/VAP x100) and linearity (LIN, VSL/VCL

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x100) were evaluated by a computer sperm cell analysis system (CASA, IVOS Animal

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Version 12.3D, Hamilton-Thorn, Inc., Beverly, MA). At each assessment, 15 µL of semen

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was placed on a pre-warmed Cell-Vu® slide (Millenium Sciences, Inc., Waltham, MA) with

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two counting chambers and >800 sperm were evaluated at 40X magnification. The settings

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for CASA were frame acquired, 20; acquisition rate, 50 Hz; minimum contrast, 45; minimun

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cell size, 4; threshold straightness, 80; medium velocity cut off, 25  µm/s; low medium

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velocity cut off, 24.9  µm/s; non motile head size, 13; non motile head intensity, 25; static

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size limits, 0.53–3.40; static intensity limits, 0.26–3.36; static elongation limits, 12–80 [2].

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2.3 Sperm morphology Sperm morphology was evaluated using eosin-nigrosin staining. A 20 µL of sperm

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sample was mixed on a slide with 20 µL of stain (Hancock Stain®, Animal Reproduction

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Systems Inc., Chino, CA). The mixture was smeared on a slide and dried on a warming plate

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at 37°C to avoid hypotonic effects. On each slide, 200 sperm were examined with a bright

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field microscope at X1000 magnification and classified as normal or with morphological

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defects in head, midpiece and tail. Results are expressed as percentages of morphologically

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normal sperm per total sperm [26].

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2.4 Sperm viability Percentages of viable sperm were assessed using LIVE/DEAD Sperm Viability Kit

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(Molecular Probes, Eugene, OR) for flow cytometry. Samples were extended ~1 x106

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sperm/mL in HEPES-buffered saline (HBS: 20mM HEPES, 137mM NaCl, 10mM glucose,

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2.0mM KOH, pH 7.4), stained with 100nM SYBR® and 10 mM propidium iodine (PI) as

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final concentrations, and incubated at 37°C for 10 min in the dark before assessment. Sperm

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exhibiting SYBR―/PI― were considered as viable sperm [27].

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The flow cytometric analyses were performed with a BD FACSCaliburTM flow cytometer

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and BD CellQuestTM Pro (BD Biosciences, San Jose, CA). This instrument was equipped

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with a 488-nm blue laser and a 640-nm diode red laser. The filters were 530/30 nm band pass

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for green fluorescence and 670 nm/long pass band for red fluorescence. For each sample, a

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minimum of 15000 events was assessed. Unstained and single-stained samples for each

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fluorochrome were used as controls and to set compensation.

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2.5 Hypoosmotic swelling test The membrane functionality was evaluated by hypoosmotic swelling (HOS) test. A

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100 µL aliquot of sperm sample was extended in 1 mL of hypoosmotic solution (150 mOsm

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sucrose; Millipore Sigma) and incubated at 37°C for 30 min. Two hundred sperm were

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evaluated under contrast field microscope at X 400 magnification. Sperm with unaltered tail

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morphology were considered HOS–, and sperm with a distinctive curling of the tail were

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considered HOS+. Results are expressed as percentages of HOS+ sperm per total sperm [28].

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2.6 Statistical analysis Statistical analyses were performed using R Core Team (2018) Version 3.5.1. (R: A

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language and environment for statistical computing; R Foundation for Statistical Computing,

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Vienna, Austria). Sperm parameters were examined by two-factor repeated measures design

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with treatment as the between-factor, incubation as the within-factor and stallion as a random

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effect for repeated measures. A mixed model was fit to each sperm parameter separately.

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Tukey adjusted pairwise comparisons were considered among treatments and incubation time

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after cooling. Results are presented as mean ± SEM. Statistical differences were considered to

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occur at P < 0.05.

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3. Results

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3.1 Sperm motility parameters

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As main effects, the addition of CF and PT alone or both combined and the incubation

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time after cooling affected total motility (treatment, P < 0.03; incubation, P < 0.0001) and

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sperm kinematic parameters as VAP (treatment, P < 0.08; incubation , P < 0.0001), VCL

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(treatment, P < 0.02; incubation , P < 0.0001) and ALH (treatment, P < 0.04; incubation , P <

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0.02). Treatment and incubation as main affects did not influence STR and LIN (P > 0.2). No

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significant interaction was observed between treatment and incubation time for motility and

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each sperm kinematic parameter assessed. No differences on sperm motility and kinematics

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were found if the additives were supplemented prior or after cooling (P > 0.1). Within

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treatment, the addition of PT after cooling reduced total motility significantly during

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incubation, but with higher values at 30 min (Table 1). Sperm treated with both CFPT before

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cooling or added separately, CF and PT, after cooling showed a significant decreasing of

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VAP values during incubation. Similarly, sperm incubated with CFPT before cooling or

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added separately after cooling displayed a significant decreasing of VCL during incubation.

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Between treatments, we noticed that sperm supplemented with PT alone or plus CF after

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cooling, displayed higher VCL values at 30 min incubation when compared to sperm treated

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with CFPT before cooling, but it did not differ from sperm incubated with CF or PT

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individually, or with control. For VSL, treatment as main effect did not alter VSL values (P >

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0.9), but the incubation after cooling did (P < 0.0001). However, no significant pairwise

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differences were detected within treatment or between treatments for each incubation time-

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point. Within treatment for ALH, sperm incubated with PT added after cooling exhibited

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significantly reduced values during incubation (Table 1).

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3.2 Morphology The percentage of morphologically normal sperm was affected by the inclusion of

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additives (P < 0.0001) and incubation (P < 0.005) after cooling as main effects. An

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interaction was observed between treatments and incubation (P < 0.04). No significant

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differences were noticed within treatments. However, sperm incubated with PT added after

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cooling at 0 min of incubation had reduced percentage of morphologically normal sperm as

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well as sperm incubated with CF and PT added before cooling at 60 min of incubation (Table

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2). The percentages of head or midpiece defects were similar among treatments and

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incubation (P > 0.3). However, the percentage of tail defects was affected by treatment (P <

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0.003) and incubation (P < 0.0003), displaying a slight interaction between treatment and

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incubation (P = 0.06). Sperm incubated with CFPT added prior cooling and all sperm

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supplemented after cooling, exhibited significantly higher percentages of tail defects during

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each incubation time-points (Table 2). We noted between treatments that sperm incubated in

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control conditions had lower percentages of tail defects during incubation. Sperm

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supplemented with PT prior cooling displayed significantly higher percentages of tail defects

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compared to control or sperm treated CF before cooling and CFPT added after cooling (Table

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2).

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3.3 Sperm viability As main effect, the percentage of viable sperm was affected by the addition of CF, PT

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or both combined (P < 0.0001) and incubation (P < 0.0001), with no interaction (P > 0.9).

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For sperm incubated in control condition, the percentage of viable sperm was not affected

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(Table 3). However, for sperm treated with CF, PT and CFPT, percentage of viable sperm

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was consistently reduced during incubation for each treatment. Sperm incubated with

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additives added prior cooling had significantly lower percentages of viable sperm compared

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to control and sperm treated after cooling (Table 3).

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3.4 Membrane functionality (HOS test) As main effect, the percentage of HOS+ sperm was affected by incubation (P <

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0.0001) and with less significance by treatment (P = 0.06). No interaction was noted (P >

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0.3). Sperm from control and treatments displayed a sustained reduction of percentages of

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HOS+ sperm, except for those sperm incubated with CF added before cooling (Table 4).

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4. Discussion The effect of methylxanthine derivatives, as caffeine and pentoxifylline have been

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previously described on boar, bull, buffalo, human, ram and stallion sperm [7,9,21,29–33]. In

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this study, we found that the addition of CF and PT to the cooling extender containing stallion

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sperm did not improve motility parameters, either added prior or after the cooling period. We

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observed a decreasing in total motility, VAP, VCL and ALH values in sperm treated with PT

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alone or associated to CF, added either before or after cooling. Sperm incubated with CF also

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showed a reduction of VAP and VCL values when it was added after cooling. In line with our

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results, the addition of CF to noncapacitating and capacitating media containing fresh stallion

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sperm resulted in reduction of motility parameters as early as 30 min of incubation [34]. In

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frozen thawed stallion sperm, CF also promoted a decrease in sperm motility [35] and PT did

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not modify motility parameters under capacitating condition [23,36]. Conversely in other

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studies, the addition of PT and CF or both additives associated to stallion frozen sperm after

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thawing improved motility, kinematic parameters and sperm survival [7,8]. Also, the addition

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of PT enhanced motility in cooled stallion sperm [9]. In epididymal stallion sperm, CF and

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PT enhanced motility and kinematic parameters [35,37], and also PT improved membrane

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integrity as tyrosine phosphorylation status remained unaffected [37]. Caffeine and PT seem

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to be more efficient in epididymal sperm than in ejaculated sperm because its ability to

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increase levels of cAMP appears to be more effective on quiescent sperm compared to

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already motile sperm [35].

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In other species, CF and PT did not stimulate motility in bull sperm, but modified

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kinematic parameters after 60 min incubation [33]. In ram, buffalo and human sperm, the

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adding of CF and PT to cooled or frozen-thawed sperm had a positive impact on motility

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during the first 30 min [32,24,38,39]. In the other hand, donkey sperm seems to not response

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to CF and PT inclusion [8]. The sperm response on motility to the exposure of CF and PT can

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differ due to specie-specific differences, type of sperm (epididymal, fresh, cooled, frozen-

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thawed), use of different additive and sperm concentration, residual amount of seminal

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plasma for cooled samples and the interaction with components of the extender [7,32,40].

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The effects of methylxanthine derivatives on sperm motility appeared to be immediate

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and transitory. In the present study, sperm supplemented with PT after cooling showed an

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increase of motility, VAP and VCL. In stallion frozen-thawed sperm, motility improved after

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20 min when CF was added after thawing [35]. Previous studies in stallion sperm noted that

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sperm treated with PT had and maintained higher motility compared to control samples

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during incubation, despite of the motility decreased for all treatments [7,9]. Caffeine and PT

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increases sperm intracellular ATP levels, resulting in the intensification of sperm efforts with

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a subsequent exhaustion of energy [7,25]. This effect results in an improvement on sperm

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motility and kinematic parameters during the first 30 min of exposure to CF and PT, but

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sperm exhaustion after a long term of exposure occurs with a potential detrimental effect.

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In the present study, we observed a significant reduction for percentages of

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morphologically normal sperm in samples supplemented with CF or PT added prior cooling.

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We found that the decrease in normal morphology was originated for an increasing of tail

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defects, effect associated to presence of CF and PT and incubation time. A previous study

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reported that CF and PT did not exhibit negative effect on frozen-thawed stallion sperm [40].

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However, sperm tail abnormalities can result in a failure in the energy provisions and motility

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defects. Alteration of some sperm tail fibrous sheath constituting proteins has been linked to

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reduced glycolysis, sperm energy production and sperm motility, resulting in a reduction of

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sperm fertility potential [41].

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We found that sperm viability was reduced during incubation in presence of CF and

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PT, and this effect was more notorious in sperm treated with CF or PT before cooling.

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Additionally, the membrane functionality as measured by HOS test was reduced during

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incubation, without a significant influence of the inclusion of CF or PT into extender. Similar

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findings have been seen in human and stallion sperm as exposure to CF and PT was

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unfavorable to maintain sperm motility and viability [30,35,25,42]. Recently, it has been

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reported that stallion sperm held in noncapacitating and capacitating media with CF impaired

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viability and acrosome integrity after 30 min incubation, whereas membrane fluidity was not

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affected by CF but increased with incubation time [34]. Caffeine increases tyrosine

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phosphorylation level and acrosomal exocytosis induced by lysophosphatidylcholine [43].

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Likewise, pentoxifylline induces a transient increasing of membrane mitochondrial activity in

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bull sperm [33] and increases the proportion of live-capacitated stallion sperm [23]. These

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findings suggest that the prolonged exposure of sperm to CF and PT has detrimental effect on

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sperm membrane integrity, reducing the proportion of viable and functional sperm and

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inducing capacitation-like changes that can comprise the sperm fertility potential in vivo

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conditions.

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5. Conclusions Despite of a promising result in previous studies, the addition of caffeine or

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pentoxifylline to cooled stallion sperm seems to be not conclusive. We evaluated the effect of

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caffeine and pentoxifylline on stallion sperm added prior and after a 24 h holding period at

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4°C. We concluded that only pentoxifylline promoted an improvement of motility and

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kinematic parameters, but just for a short time when added to stallion sperm after cooling. On

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the other hand, normal sperm morphology and sperm membrane integrity were impaired after

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a prolonged exposure to caffeine or pentoxifylline, potentially compromising the fertility

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potential by exhaustion of energy and capacitation-like changes. Further research is needed to

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evaluate the effects of these additives under standardized conditions for reagent and sperm

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concentration; interactions between additives and extender components; and the outcome of

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artificial insemination of mares with supplemented cooled semen.

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Author declaration of interests

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The authors declare no conflict of interest.

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Funding

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This work was supported by the Università di Padova-Ministry of the University and of

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Scientific and Technologic Research (MURST, Italy) - Ex 60%.

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Acknowledgements

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The authors wish to thank Dr. Stefani A.L. and her staff at the Istituto Zooprofilattico

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Sperimentale delle Venezia (IZSVe) for the help in the flow cytometry analysis.

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Authorship

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All authors defined the study design. M. Rossi and M.E. Falomo executed the study. R.

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Gonzalez-Castro analyzed the data. M. Rossi and R. Gonzalez-Castro prepared the

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manuscript, and all authors read and approved the final manuscript.

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Ethical Issues

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According to the Italian law for the protection of experimental animals (Law Decree n. 26

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issued on 4 March 2014, art. 2), the approval by an ethical committee is not required under

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the circumstances that this trial was carried out.

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Table 1. Mean ± SEM of percentage of total motile sperm and kinematic sperm parameters by treatment and incubation time point after 24 h cooling at 4°C. Caffeine (CF), pentoxifylline (PT) or both combined (CFPT) were added to stallion sperm before or after cooling. Control contained no additives and incubated under same conditions. After cooling, sperm were incubated at 37°C for 120 min. Sperm parameter

448 449 450 451 452

Treatments

0 min

30 min

60 min

120 min

Control 67.3 ± 6.1 60.9 ± 6.1 60.4 ± 6.1 55.2 ± 6.1 CF 59.0 ± 6.1 62.1 ± 6.1 59.2 ± 6.1 49.7 ± 6.1 Precooling PT 56.3 ± 6.1 54.8 ± 6.1 53.4 ± 6.1 39.3 ± 6.1 Total CFPT 54.2 ± 6.1 50.1 ± 6.1 55.7 ± 6.1 50.6 ± 6.1 motility (%) CF 64.8 ± 6.1 63.7 ± 6.1 63.8 ± 6.1 57.4 ± 6.1 ab a ab Postcooling PT 58.2 ± 6.1 66.3 ± 6.1 51.1 ± 6.1 43.9 ± 6.1b CFPT 62.9 ± 6.1 65.9 ± 6.1 56.7 ± 6.1 46.4 ± 6.1 Control 60.7 ± 3.8 51.6 ± 3.8 55.6 ± 3.8 50.6 ± 3.8 CF 59.8 ± 3.8 52.4 ± 3.8 56.2 ± 3.8 49.2 ± 3.8 Precooling PT 56.8 ± 3.8 50.1 ± 3.8 52.5 ± 3.8 47.8 ± 3.8 VAP CFPT 58.9 ± 3.8a 50.0 ± 3.8ab 49.6 ± 3.8ab 46.9 ± 3.8b (µm/s) a ab ab CF 62.9 ± 3.8 57.9 ± 3.8 55.8 ± 3.8 48.6 ± 3.8b Postcooling PT 57.0 ± 3.8a 59.3 ± 3.8ab 52.4 ± 3.8ab 46.9 ± 3.8b CFPT 61.0 ± 3.8 60.8 ± 3.8 52.9 ± 3.8 51.6 ± 3.8 a abAB ab Control 138.5 ± 7.6 119.7 ± 7.6 120.6 ± 7.6 114.7 ± 7.6b AB CF 134.8 ± 7.6 123.1 ± 7.6 121.9 ± 7.6 114.8 ± 7.6 Precooling AB PT 128.3 ± 7.6 111.9± 7.6 116.1± 7.6 112.3± 7.6 VCL a bB ab CFPT 128.6 ± 7.6 107.2 ± 7.6 112.2 ± 7.6 107.4 ± 7.6b (µm/s) CF 139.4 ± 7.6a 126.4 ± 7.6abAB 123.9 ± 7.6ab 112.6 ± 7.6b Postcooling PT 127.6 ± 7.6a 132.9 ± 7.6aA 119.6 ± 7.6ab 106.3 ± 7.6b CFPT 133.4 ± 7.6 132.9 ± 7.6A 120.3 ± 7.6 116.4 ± 7.6 Control 38.6 ± 2.9 31.4 ± 2.9 32.3 ± 2.9 32.8 ± 2.9 CF 36.4 ± 2.9 31.9 ± 2.9 36.8 ± 2.9 29.0 ± 2.9 Precooling PT 35.7 ± 2.9 30.0 ± 2.9 33.1 ± 2.9 30.0 ± 2.9 VSL CFPT 38.8 ± 2.9 31.0 ± 2.9 33.3 ± 2.9 30.6 ± 2.9 (µm/s) CF 38.3 ± 2.9 34.3 ± 2.9 34.8 ± 2.9 29.8 ± 2.9 Postcooling PT 35.3 ± 2.9 37.6 ± 2.9 32.0 ± 2.9 29.6 ± 2.9 CFPT 34.5 ± 2.9 36.7 ± 2.9 32.2 ± 2.9 32.4 ± 2.9 Control 7.1 ± 0.7 6.4 ± 0.7 6.5 ± 0.7 5.6 ± 0.7 CF 6.9 ± 0.7 6.0 ± 0.7 5.7 ± 0.7 5.2 ± 0.7 Precooling PT 6.1 ± 0.7 5.2 ± 0.7 5.3 ± 0.7 5.1 ± 0.7 ALH CFPT 6.0 ± 0.7 4.7 ± 0.7 5.3 ± 0.7 5.4 ± 0.7 (µm) CF 7.0 ± 0.7 6.7 ± 0.7 7.0 ± 0.7 6.1 ± 0.7 Postcooling PT 7.2 ± 0.7a 6.4 ± 0.7ab 4.6 ± 0.7b 6.1 ± 0.7ab CFPT 6.2 ± 0.7 6.1 ± 0.7 5.6 ± 0.7 6.4 ± 0.7 VAP: average path velocity. VCL: curvilinear velocity. VSL: straight-linear velocity. ALH: amplitude of lateral head displacement. a,b Rows means within treatments, different superscripts are different at P < 0.05. A,B Column means within incubation and each sperm parameter, different superscripts are different at P < 0.05.

20

453 454 455 456 457 458

Table 2. Mean ± SEM of percentage of morphologically normal sperm and tail defects by treatment and incubation time point after 24 h cooling at 4°C. Caffeine (CF), pentoxifylline (PT) or both combined (CFPT) were added to stallion sperm before or after cooling. Control contained no additives and incubated under same conditions. After cooling, sperm were incubated at 37°C for 120 min. Sperm parameter

459 460 461 462 463

Treatments

0 min

30 min

60 min

120 min

68.4 ± 2.6A 66.0 ± 2.6 66.3 ± 2.6A 64.8 ± 2.6 Control 65.0 ± 2.6AB 59.2 ± 2.6 58.3 ± 2.6B 62.1 ± 2.6 CF Precooling 58.9 ± 2.6B 58.9 ± 2.6 63.2 ± 2.6AB 63.2 ± 2.6 PT Morphologically 64.0 ± 2.6AB 64.0 ± 2.6 59.6 ± 2.6AB 59.6 ± 2.6 CFPT normal sperm (%) 59.1 ± 2.6AB 61.4 ± 2.6 59.1 ± 2.6AB 60.0 ± 2.6 CF 58.1 ± 2.6B 59.8 ± 2.6 59.8 ± 2.6AB 58.1 ± 2.6 Postcooling PT 59.6 ± 2.6AB 60.3 ± 2.6 64.8 ± 2.6AB 65.2 ± 2.6 CFPT 7.3 ± 1.2A 7.8 ± 1.2 7.7 ± 1.2 7.7 ± 1.2 Control A 6.8 ± 1.2 8.8 ± 1.2 9.5 ± 1.2 10.2 ± 1.2 CF Precooling B 13.4 ± 1.2 10.6 ± 1.2 10.7 ± 1.2 11.4 ± 1.2 PT Tail defects aA a ab 6.4 ± 1.2 8.2 ± 1.2 9.0 ± 1.2 12.1 ± 1.2b CFPT (%) 10.2 ± 1.2abAB 7.0 ± 1.2a 10.8 ± 1.2ab 12.2 ± 1.2b CF 10.2 ± 1.2abAB 6.8 ± 1.2b 11.6 ± 1.2a 11.1 ± 1.2ab Postcooling PT 7.7 ± 1.2aA 7.0 ± 1.2a 9.5 ± 1.2ab 12.5 ± 1.2b CFPT a,b Rows means within treatments, different superscripts are different at P < 0.05. A,B Column means within incubation and each sperm parameter, different superscripts are different at P < 0.05.

21

464 465 466 467 468

Table 3. Mean ± SEM of percentage of viable sperm by treatment and incubation time point after 24 h cooling at 4°C. Caffeine (CF), pentoxifylline (PT) or both combined (CFPT) were added to stallion sperm before or after cooling. Control contained no additives and incubated under same conditions. After cooling, sperm were incubated at 37°C for 120 min. Treatments

469 470 471 472 473

0 min 30 min 60 min A A Control 55.7 ± 3.4 55.6 ± 3.4 55.2 ± 3.4A CF 45.6 ± 3.4aBCD 44.9 ± 3.4a 43.0 ± 3.4abBC Pre-cooling PT 42.7 ± 3.4aDC 40.5 ± 3.4aC 36.9 ± 3.4aC 36.1 ± 3.4abC 36.0 ± 3.4abC CFPT 38.4 ± 3.4aD CF 50.0 ± 3.4aABC 50.4 ± 3.4aAB 48.3 ± 3.4aBC Post-cooling PT 55.1 ± 3.4aABC 52.8 ± 3.4aA 51.6 ± 3.4aA 52.2 ± 3.4aAB 48.7 ± 3.4aBC CFPT 52.4 ± 3.4aAB a,b Rows means within treatments, different superscripts are different at P < 0.05. A,B Column means within incubation, different superscripts are different at P < 0.05.

120 min 49.1 ± 3.4A 35.8 ± 3.4bBC 27.8 ± 3.4bC 29.0 ± 3.4bC 40.0 ± 3.4bAB 40.5 ± 3.4bAB 38.4 ± 3.4bB

22

474 475 476 477 478

Table 4. Mean ± SEM of percentage of HOS+ sperm by treatment and incubation time point after 24 h cooling at 4°C. Caffeine (CF), pentoxifylline (PT) or both combined (CFPT) were added to stallion sperm before or after cooling. Control contained no additives and incubated under same conditions. After cooling, sperm were incubated at 37°C for 120 min. Treatments

479 480 481

0 min 30 min 60 min a a 37.3 ± 3.1 34.9 ± 3.1 33.6 ± 3.1ab Control 34.5 ± 3.1 35.5 ± 3.1 31.0 ± 3.1 CF a ab 42.4 ± 3.1 31.7 ± 3.1 29.9 ± 3.1ab Pre-cooling PT 38.8 ± 3.1a 35.1 ± 3.1ab 36.4 ± 3.1ab CFPT 32.8 ± 3.1b 37.0 ± 3.1ab 41.2 ± 3.1a CF 40.7 ± 3.1a 34.6 ± 3.1ab 31.9 ± 3.1b Post-cooling PT 41.6 ± 3.1a 35.1 ± 3.1ab 33.4 ± 3.1b CFPT a,b Rows means within treatments, different superscripts are different at P < 0.05.

120 min 27.0 ± 3.1b 34.4 ± 3.1 28.6 ± 3.1b 29.6 ± 3.1b 31.4 ± 3.1b 30.7 ± 3.1b 32.8 ± 3.1b

23

Highlights

Effect of caffeine and pentoxifylline added before or after cooling on sperm characteristics of stallion sperm Melissa Rossia*, Raul Gonzalez-Castrob, Maria Elena Falomoc

•

Pentoxifylline added after cooling promoted short-term sperm motility.

•

Caffeine and pentoxifylline increased sperm tail deffects.

•

Sperm viablity and membrane intergrity was impaired by caffeine and pentoxifylline.

•

Both additives boost short-term motility but are detrimental in long-term exposure.

UNIVERSITÀ DEGLI STUDI DI PADOVA

Legnaro (PD), 31st October 2019

Dear Editor, Biological samples used in the article entitled "Effect of caffeine and pentoxifylline added before or after cooling on sperm characteristics of stallion sperm" are part of routinely procedures not performed for experimental purposes. According to the Italian law for the protection of experimental animals (Law Decree n. 26 issued on 4 March 2014, art. 2), the approval by an ethical committee is not required under the circumstances that this trial was carried out.

Kind regards, Melissa Rossi, DVM, PhD Raul Gonzalez-Castro, DVM, PhD Maria Elena Falomo, DVM, PhD

UNIVERSITÀ DEGLI STUDI DI PADOVA

Legnaro (PD), 31st October 2019

Dear Editor, We wish to confirm that there are no known conflicts of interest and no significant financial support that could have influenced the outcome of the article entitled "Effect of caffeine and pentoxifylline added before or after cooling on sperm characteristics of stallion sperm" and proposed to Journal of Equine Veterinary Science. Kind regards, Melissa Rossi, DVM, PhD Raul Gonzalez-Castro, DVM, PhD Maria Elena Falomo, DVM, PhD