Preservation of Epididymal Stallion Sperm in Liquid and Frozen States: Effects of Seminal Plasma on Sperm Function and Fertility

Journal Pre-proof Preservation of epididymal stallion sperm in liquid and frozen states: effects of seminal plasma on sperm function and fertility Jordi Miró, Roser Morató, Ingrid Vilagran, Ester Taberner, Sergi Bonet, Marc Yeste PII:

S0737-0806(20)30031-9

DOI:

https://doi.org/10.1016/j.jevs.2020.102940

Reference:

YJEVS 102940

To appear in:

Journal of Equine Veterinary Science

Received Date: 31 July 2019 Revised Date:

11 November 2019

Accepted Date: 22 January 2020

Please cite this article as: Miró J, Morató R, Vilagran I, Taberner E, Bonet S, Yeste M, Preservation of epididymal stallion sperm in liquid and frozen states: effects of seminal plasma on sperm function and fertility, Journal of Equine Veterinary Science (2020), doi: https://doi.org/10.1016/j.jevs.2020.102940. 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. © 2020 Elsevier Inc. All rights reserved.

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Title

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Preservation of epididymal stallion sperm in liquid and frozen states: effects of seminal

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plasma on sperm function and fertility.

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Authors

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Jordi Miró1, *, Roser Morató1, 2; Ingrid Vilagran2; Ester Taberner1; Sergi Bonet2; Marc Yeste2

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Affiliations

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1

Equine Reproduction Service, Department of Animal Medicine and Surgery, Faculty of

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Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra (Barcelona),

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Spain.

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Institute of Food and Agricultural Technology, University of Girona, E-17071 Girona, Spain.

Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology,

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*Corresponding author

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Jordi Miró, Equine Reproduction Service, Department of Animal Medicine and Surgery,

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Faculty of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra

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(Barcelona), Spain.< [email protected]> Phone: +34 935814273; Fax: +34 935811044

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Abstract

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Three separate experiments were conducted to improve preservation of stallion epididymal

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sperm. In the first one, two different cooling extenders (Kenney and Gent) were compared.

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Sperm viability and motility patterns were assessed in 10 different epididymal sperm samples

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after 0 h, 24 h, 48 h, 72 h and 96 h of preservation at 4ºC. No significant differences were

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observed in any of the evaluated parameters either between extenders or throughout the

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storage period. The second set of experiments was designed to determine whether

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supplementing thawing medium (INRA-Freeze™) with seminal plasma had any impact on the

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quality of frozen-thawed epididymal sperm. Ten epididymal frozen-thawed sperm samples

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coming from separate stallions were used and different functional parameters (sperm

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membrane integrity and lipid disorder, motility, intracellular Ca2+ levels, and intracellular

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concentrations of peroxides and superoxides) were evaluated after incubation with or without

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50% seminal plasma. Supplementing thawing medium with seminal plasma had no impact on

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sperm function and survival. The third experiment was an in vivo study. Twenty-five mares

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were inseminated with epididymal frozen-thawed sperm and seminal plasma, and 21 were

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bred with epididymal frozen-thawed sperm only. Pregnancy rates obtained for mares

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artificially inseminated with epididymal frozen-thawed sperm and seminal plasma were

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significantly (P<0.05) higher than those observed when seminal plasma was not infused (64%

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vs. 19%). Taken together, our data indicate that the quality of epididymal stallion sperm can

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be maintained at 4ºC for up to 96 hours. In addition, not only does supplementing frozen-

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thawed epididymal sperm with seminal plasma have any damaging effect on their quality, but

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it may also improve pregnancy rates after artificial insemination.

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Keywords: equine; epididymal sperm; preservation; seminal plasma; artificial insemination.

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1. Introduction

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Obtaining epididymal sperm from dead or recently castrated stallions and from males

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with acquired genital tract obstructions (which are unable to ejaculate) opens up a way to

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preserve the genetic material of high-value animals. In this context, although cryopreserved

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epididymal sperm may allow storing valuable genetics of a given stallion after unforeseen

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death or injury [1], there is controversy on whether or not epididymal stallion sperm may be

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preserved at 4°C [2]. While several works have demonstrated that epididymal stallion sperm

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may be kept within the epididymides and maintained at 4ºC prior to cryopreservation [3-7],

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storage of epididymal sperm rather than that of the whole gonad has been less studied.

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Related with this, one should note that successful preservation of epididymal sperm at 4°C

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could make this technology more practical and available for its use, and would decrease

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transport costs. In addition, the use of the egg- and milk-based extenders that are currently

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used for ejaculated semen could also contribute to the preservation of epididymal sperm. In

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fact, although pregnancies following artificial insemination (AI) with epididymal stallion

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sperm [5-7] have been observed, only few studies have focused on whether epididymal sperm

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may be preserved using a milk-based extender containing egg yolk and have evaluated the

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impact of adding antioxidants, such as D-penicillamine [24].

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Apart from liquid storage at 4°C, cryopreservation is another strategy for preserving

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stallion sperm. However, while freeze-thawing of stallion epididymal sperm is currently

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possible [1,3,8-16] and may give birth to healthy foals [1,3,6,9-15], there is controversy on

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pregnancy rates. In effect, on the one hand, Monteiro et al (2011) [5] found that viability and

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fertilizing ability of cauda epididymal sperm (cooled and frozen-thawed) are similar to those

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of ejaculated semen, and Neuhauser et al (2019) [15] compared five extenders and

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demonstrated that freezing media containing low concentrations of glycerol, either with or

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without methylformamide, were the ones that showed the highest post-thaw epididymal sperm

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quality. On the other hand, other studies concurred that fertilization rates are higher with

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ejaculated semen (cooled and frozen-thawed) than with epididymal sperm [7, 17]. These

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controversies make preservation of epididymal stallion sperm to be considered as highly

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challenging and warrant further research.

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Supplementing frozen-thawed epididymal stallion sperm with seminal plasma could

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improve their post-thaw quality and fertilizing ability. While seminal plasma is known to

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affect sperm physiology, its effects rely upon species and the nature of the sample.

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Furthermore, seminal plasma has been reported to inhibit sperm binding to neutrophils in

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horses and donkeys, and this has been suggested to yield better reproductive performances

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[18-20]. However, the effects of seminal plasma on the motility of fresh/cooled and frozen-

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thawed epididymal sperm are inconsistent across the literature. Indeed, while most previous

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studies observed that seminal plasma has a beneficial effect on freshly harvested epididymal

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spermatozoa [1, 14, 21], others reported that the effects of seminal plasma on post-thaw

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sperm quality are less clear. For example, Neuhauser et al. [14] found that whereas adding

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20% or 50% of seminal plasma increased the motility of epididymal spermatozoa in 10

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stallions, it had no effect on the sperm motility of six stallions, regardless of the concentration

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of seminal plasma.

87

Against this background, the present study sought to determine whether: a) epididymal

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sperm can be stored at 4°C for up to 96 h using two commercial extenders available for

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ejaculated semen (Kenney and Gent); b) supplementing frozen-thawed epididymal stallion

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sperm with seminal plasma has any impact on their quality; and c) intrauterine infusion of

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seminal plasma just after AI with frozen-thawed epididymal stallion sperm has any effect on

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fertility rates.

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2. Materials and methods

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2.1. Experimental design

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This work consisted of three separate experiments. The aim of the first experiment was

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to determine whether two commercial extenders (Kenney and Gent) are able to preserve

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epididymal stallion spermatozoa at 4ºC up to 96 hours. With this purpose, epididymal sperm

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samples from 10 stallions were collected and divided into two groups (diluted in Kenney or

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Gent extenders) and then stored at 4°C for 96 h. Sperm motility and viability were evaluated

100

after semen collection (0 h), and after 24, 48, 72 and 96 h of cooled storage. We also

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compared sperm motile subpopulations between these two extenders (Table 4).

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The second study was conducted to determine the effects of adding seminal plasma to

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frozen-thawed epididymal spermatozoa . Samples were cryopreserved and stored for 2-5

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years. Upon thawing, the content of straws was diluted to a final concentration of

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100×106·spermatozoa/mL with Beltsville Thawing Solution (BTS), and supplemented with

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0% or 50% of seminal plasma. Samples were incubated at 37ºC for 15 min, prior to

107

evaluating sperm motility, viability, membrane lipid disorder, and intracellular levels of Ca2+

108

levels, peroxides and superoxides.

109

The third study analyzed the effects of adding seminal plasma to epididymal frozen-

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thawed sperm on fertility rates. The study included 46 mares (aged 4-10 years old) that

111

showed good body condition and were known to be fertile. Mares were divided into two

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groups: a) 21 mares were inseminated with epididymal frozen-thawed sperm (control); and b)

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25 mares were inseminated with frozen-thawed epididymal sperm prior to infusing seminal

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plasma into the uterine body.

115 116

2.2. Reagents and media

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All chemicals and reagents were purchased from Sigma-Aldrich (Saint Louis, MO,

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USA) unless otherwise stated. All fluorochromes (Fluo-3-AM, Merocyanine 540, propidium

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iodide (PI), 2’, 7’-diclorodihydrofluorescein diacetate (H2DCFDA), hydroethidine (HE),

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SYBR14 and YO-PRO-1) were purchased from Invitrogen Molecular Probes (Termofisher

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Scientific; Waltham, MA, USA). Stock solutions of fluorescence probes were prepared in

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DMSO at a final concentration of 1 mM, except YO-PRO-1 (25 µM), and PI (1 mg/mL in

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water). These stocks were kept at -20 ºC in the dark. Flow cytometry equipment, software,

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and consumables were purchased from Beckman Coulter (Beckman Coulter Inc.; Brea, CA,

125

USA).

126

Kenney semen extender was made in our laboratory, Gent extender was purchased

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from Minitüb (Tiefenbach, Germany) and INRA Freeze ™ (2.5% glycerol) was provided by

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IMV Technologies (L’Aigle, Cedex, France).

129 130

2.3. Harvesting of epididymal spermatozoa

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All animals attended the Veterinary Hospital, Autonomous University of Barcelona

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(Bellaterra, Cerdanyola del Vallès, Spain). Sperm samples was collected from the epididymes

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of 10 mature stallions, aged 3-7 years old, after animal castration (n=9) or euthanasia (n=1).

134

Stallions were castrated under general anesthesia by intratesticular injection of 10 mL

135

lidocaine, which is known to have no impact on epididymal sperm quality [22]. One stallion

136

was euthanized just after a femur fracture.

137

Testis-epididymis complexes were transported in insulated containers at 20°C to the

138

laboratory (transport time <10 min). Upon arrival at the laboratory, each epididymis was

139

dissected from the testis, and the tail and ductus deferens were isolated. Sperm were collected

140

using the retrograde flow technique [17]. Vas deferens and epididymal cauda were dissected

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free of the fascia, working distally from the vas deferens towards the epididymal corpus. A

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syringe containing 10 mL of Kenney extender with a blunt, 20-gauge needle was used to flush

143

one epididymis. The contralateral epididymis was washed using a syringe containing 10 mL

144

of Gent egg-yolk extender. Both extenders had been preheated to 37ºC. Afterwards, flushing

145

was recovered from each epididymis in a 15-mL conical tube, adjusted to a final

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concentration of 100×106 viable spermatozoa/mL, and incubated in a water bath for 15 min.

147

Sperm concentration was evaluated using a Neubauer chamber and sperm viability through

148

eosin-nigrosin staining.

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2.4. Collection and preparation of seminal plasma samples

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Five ejaculates from three stallions were collected through a Hannover artificial

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vagina (Minitüb) with an in liner nylon filter. These stallions were healthy, yielded semen of

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good quality both before and after freeze-thawing, and were of proven fertility.

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Seminal plasma was obtained after double semen centrifugation (800×g for 10 min at

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25 ºC), followed by filtration through membranes with a 0.22-µm pore. The absence of

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spermatozoa was confirmed under a phase-contrast microscope (Olympus Europe, Hamburg,

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Germany) at 200× magnification. Following this, all samples were pooled and stored at -80ºC,

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until used. Thawing of seminal plasma was performed at 37ºC in a water bath.

159 160

2.5. Preservation of epididymal stallion sperm at 4ºC

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Epididymal stallion sperm were stored at 4ºC for 96 h under anaerobic conditions.

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After 24, 48, 72 and 96 h of storage at 4ºC, an aliquot of 1 mL was taken and incubated in a

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water bath at 37ºC for 15 min, prior to evaluating sperm viability and motility.

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2.6. Cryopreservation of epididymal sperm

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Cryopreservation of sperm cells was performed as described by Yeste et al. [23] with

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slight modifications. Briefly, the remaining epididymal sperm flushed with Kenney extender

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(and not used for liquid-storage at 4ºC) was cryopreserved. Samples were placed in conical

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tubes and then centrifuged at 600×g and 20ºC for 15 min (Medifriger BL-S centrifuge; JP

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Selecta S.A., Barcelona, Spain). Supernatants were discarded and pellets were resuspended in

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a commercial freezing extender (INRA-Freeze; INRA, Paris, France) to a final concentration

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of 200×106 progressively motile spermatozoa per mL. Samples were then packaged into 0.5-

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mL straws, and frozen using an Ice-Cube 14S programmable freezer (Minitüb). The

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cooling/freezing program consisted of three steps with the following cooling/freezing rates:

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(i) from 20ºC to 5ºC for 60 min, at a rate of -0.25ºC/min; (ii) from 5 to -90 for 20 min, at a

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rate of -4.75ºC/min; and (iii) from -90ºC to -120ºC for 2.7 min, at a rate of -11ºC/min. Straws

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were finally plunged into liquid nitrogen and stored until thawing. For thawing, two straws

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per stallion were thawed by shaking in a water bath at 37ºC for 20 sec. After thawing, frozen-

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thawed epididymal sperm were incubated for 10 min at 37ºC, prior to evaluating their

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viability and motility.

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2.7. Analysis of sperm motility

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Sperm motility and kinematic parameters were determined using a computer-assisted

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sperm analysis (CASA) system (Integrated Sperm Analysis System V1.0; Proiser SL,

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Valencia, Spain). A 5-µL drop was placed on a Makler counting chamber (Sefi-Medical

186

Instruments, Israel) previously warmed to 37ºC. Samples were then observed under a phase-

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contrast microscope equipped with a heat stage (37ºC; Olympus). Three fields per drop were

188

analyzed, and the sperm kinematic parameters shown in Table 1 recorded. Total motility was

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defined as the percentage of spermatozoa with VAP of >10 µm/s, and progressive motility as

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the percentage of spermatozoa with STR of >75%.

191 192

2.8. Flow cytometry analyses

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Evaluation of sperm viability, membrane lipid disorder and intracellular levels of

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calcium, peroxides and superoxides in Experiment 2 was performed using a Cell Lab

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QuantaSC™ cytometer (Beckman Coulter; Fullerton, California, USA), as previously

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described [23]. Prior to staining, sperm concentration was adjusted to 1×106 spermatozoa/mL

197

in a final volume of 0.5 mL with HEPES-buffered saline solution (10 mM HEPES, 150 mM

198

NaCl, 10% BSA; pH = 7.4). After staining with appropriate fluorochromes, spermatozoa were

199

excited with an argon ion laser (488 nm) set at a power of 22 mW. Two optical filters (FL1

200

and FL3) were used and their technical settings were as follows: FL1 (green fluorescence):

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Dichroic/Splitter, DRLP: 550 nm, BP filter: 525 nm; and FL3 (red fluorescence): LP filter:

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670/730 nm. Signals were logarithmically amplified and photomultiplier settings were

203

adjusted to particular staining methods. Sheath flow rate was set at 4.17 µL/min and EV and

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side scatter (SS) were recorded in a linear mode (in EV vs. SS dot plots) for a minimum of

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10,000 events per replicate. At least two replicates per stallion and sperm parameter were

206

evaluated. The analyzer threshold was adjusted on the EV channel to exclude subcellular

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debris and cell aggregates, and the sperm-specific events were positively gated on the basis of

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EV/SS distributions. Calibration of this device was made periodically through 10-µm Flow-

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Check fluorospheres (beads; Beckman Coulter), the bead size being positioned at channel 200

210

on the volume scale. In some protocols, compensation was used to minimize spill-over of

211

green fluorescence into the red channel. Information on the events was collected in List-mode

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Data files (.LMD), and files were subsequently analyzed through the Cell Lab Quanta®SC

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MPL Analysis Software (version 1.0; Beckman Coulter). In all cases except for SYBR14/PI

214

staining, data were corrected following the procedure described in Yeste et al. [23].

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2.8.1. Sperm viability (SYBR14/PI)

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Sperm viability was evaluated as plasma membrane integrity through SYBR14/PI

218

staining. Samples were incubated at 37.5ºC for 10 min with SYBR14 (final concentration:

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100 nM), and then with PI (final concentration: 12 µM) at the same temperature for further 5

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min. After this assessment, samples were analyzed and spermatozoa were classified as viable

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(SYBR14+/PI-) or non-viable (SYBR14-/PI+ and SYBR14+/PI+). Debris particles (SYBR14-

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/PI-) were used to adjust the other stainings. Spill over of SYBR14 (FL1) into the PI-channel

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(FL3) was compensated (2.45%).

224 225

2.8.2. Membrane lipid disorder (M540/YO-PRO-1)

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Membrane lipid disorder was assessed through co-staining with Merocyanine 540

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(M540) and YO-PRO-1. M540 binds preferentially to membranes with loosely packed lipids,

228

whereas YO-PRO-1 stains the nuclei of cells with increased plasma membrane permeability.

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Samples were incubated with 400 µM M540 and 25 nM YO-PRO-1 for 10 min at 37.5ºC in

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the dark. Spermatozoa were classified into four categories: (i) viable spermatozoa with low

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membrane lipid disorder (M540-/YO-PRO-1-); (ii) viable spermatozoa with high membrane

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lipid disorder (M540+/YO-PRO-1-); (iii) non-viable spermatozoa with low membrane lipid

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disorder (M540-/YO-PRO-1+); and (iv) non-viable spermatozoa with high membrane lipid

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disorder (M540+/YO-PRO-1+). Data were not compensated.

235 236

2.8.3. Intracellular Ca2+levels (Fluo3/PI)

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Intracellular calcium (Ca2+) levels were evaluated together with plasma membrane

238

integrity by combining PI and Fluo3, a probe that accumulates intracellularly and increases its

239

green fluorescence when binding Ca2+. Samples were incubated with 1 µM Fluo3-AM and 12

240

µM PI for 10 min at 37.5ºC in the dark. Four populations were identified: (i) viable

241

spermatozoa with low levels of intracellular calcium (Fluo3-/PI-); (ii) viable spermatozoa with

242

high levels of intracellular calcium (Fluo3+/PI-); (iii) non-viable spermatozoa with low levels

243

of intracellular calcium (Fluo3-/PI+); and (iv) non-viable spermatozoa with high levels of

244

intracellular calcium (Fluo3+/PI+). Fluo3-AM spill over into the PI channel (2.45%) and PI

245

spill over into the Fluo3 channel (28.72%).

246 247 248

2.8.4. Intracellular peroxide levels (H2DCFDA/PI) Intracellular

levels

of

peroxides

were

determined

with

2’,7’-

249

dichlorodihydrofluorescein diacetate (H2DCFDA), which is oxidized to dichlorofluorescein

250

(DCF+) that emits fluorescence at 530 nm. This fluorescent probe was combined with PI for

251

simultaneous evaluation of sperm viability. Samples were incubated with 140 µM H2DCFDA

252

and 12 µM PI at room temperature in the dark for 60 min. Spermatozoa were classified into

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four populations: (i) viable spermatozoa with low intracellular peroxide levels (DCF-/PI-); (ii)

254

viable spermatozoa with high intracellular peroxide levels (DCF+/PI-); (iii) non-viable

255

spermatozoa with low intracellular peroxide levels (DCF-/PI+); and (iv) non-viable

256

spermatozoa with high intracellular peroxide levels (DCF+/PI+). Data were not compensated.

257 258

2.8.5. Intracellular superoxide levels (HE/YO-PRO-1)

259

Hydroethidine (HE) was used to detect intracellular superoxide levels, since HE is

260

oxidized to ethidium (E+) when superoxide anions are present. This fluorescent probe was

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combined with YO-PRO-1 for simultaneous evaluation of sperm viability. Samples were

262

incubated with 4 µM HE and 25 nM YO-PRO-1 at room temperature in the dark for 40 min.

263

Spermatozoa were classified as: (i) viable spermatozoa with low superoxide levels (E-/YO-

264

PRO-1-); (ii) viable spermatozoa with high superoxide levels (E+/YO-PRO-1-); (iii) non-

265

viable spermatozoa with low superoxide levels (E-/YO-PRO-1+); or (iv) non-viable

266

spermatozoa with high superoxide levels (E+/YO-PRO-1+). Data were not compensated.

267 268

2.9. Artificial insemination

269

Mares were checked transrectally by ultrasound three times per week. When a

270

dominant follicle reached 42 mm, in the absence of a corpus luteum and with estrus uterine

271

edema, 3,000 IU of HCG were IV administrated. Following this, mares were monitored for

272

ovulation every six hours. During or just after ovulation, mares were inseminated through

273

deep AI (utero-tubal junction) with four frozen-thawed epididymal sperm straws (for viability

274

and motility, see Table 2). Twenty-one mares were inseminated with frozen-thawed

275

epididymal sperm only (control), and 25 were inseminated with frozen-thawed epididymal

276

sperm plus seminal plasma. In the latter case, 10 mL of seminal plasma was infused into the

277

uterine body immediately after AI. Both groups of mares (i.e. inseminated with or without

278

seminal plasma) were checked to confirm ovulation at 6 h post-AI, and for the absence of

279

fluid at 12 h post-AI. Pregnancy diagnosis was performed through ultrasonography after 15

280

days of AI.

281

Each of the ten frozen-thawed epididymal sperm samples, each coming from a

282

separate stallion, was used to inseminate, at least, two mares of each group. In addition,

283

frozen-thawed epididymal sperm samples from one stallion were used to inseminate three

13

284

mares of each group, and those from four stallions were used to inseminate four mares of the

285

seminal plasma group.

286 287

2.10. Statistical analyses

288

Data were managed using SAS (SAS for Windows 9.1, SAS Institute Inc., Cary, NC,

289

USA) and SPSS statistical packages (IBM SPSS for Windows 21.0; IBM Corp, Chicago,

290

Illinois, USA).

291

In the first experiment, means and standard error of the mean of all variables were

292

determined using the PROC MEANS procedure. The FASTCLUS clustering procedure was

293

used to separate motile spermatozoa into specific subpopulations. FASTCLUS performs a

294

disjointed cluster analysis based on Euclidean distances, calculated taking into account one or

295

more quantitative variables. In this case, these variables were the different sperm kinetic

296

parameters measured by CASA. Sperm cells that shared similar motility characteristics were

297

assigned to the same cluster, whereas spermatozoa that differed in motility characteristics

298

were assigned to the other clusters. Differences between extenders and throughout the storage

299

period were determined through a mixed model (LSMEANS) followed by post-hoc Sidak’s

300

test for multiple pair-wise comparisons.

301

For the second experiment, all parameters were first checked for normality and

302

homogeneity of variances (Shapiro-Wilk and Levene tests) and then analyzed through a t-test

303

for related measures. In the third experiment, the chi-square test was used to compare fertility

304

rates between the two groups of mares. In all cases, the significance level was set at P≤0.05

305

and data are shown as means ± standard error of the mean (SEM)

306 307

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3. Results

309 310

3.1. Experiment 1

311

Tables 2 and 3 show the viability, total motility and progressive motility of epididymal

312

sperm after 0, 24, 48, 72 or 96 h of storage at 4ºC. There were no significant differences

313

between Kenney and Gent extenders either in viability or in total and progressive motilities

314

throughout the storage period.

315

Epididymal spermatozoa were also classified into four motile subpopulations, and the

316

percentages of each sperm subpopulation varied across storage at 4ºC (Table 4). As Figure 2

317

shows, subpopulation 1 (SP1), which showed low VAP, LIN, ALH and BCF decreased

318

significantly along the storage time. Subpopulation 2 (SP2), which showed medium VAP,

319

LIN, ALH and BCF slightly augmented along the storage period. SP3, which had high VAP,

320

LIN, BCF and medium ALH, also showed an increase along storage at 4ºC. SP4, which

321

presented the highest VAP, low LIN and high ALH and BCF, showed an increase after 24 h

322

of storage. We observed a tendency of sperm velocity and linearity to increase after 24 h; this

323

tendency was maintained after 48, 72 and 96 h of storage at 4ºC. Furthermore, whereas SP1

324

and SP3 tended to decrease when storage time increased, SP4 appeared to increase over that

325

storage period.

326 327

3.2. Experiment 2

328

Figures 3A and 3B show total and progressive motilities of epididymal frozen-thawed

329

sperm and Figure 3C shows the effects on the other sperm parameters evaluated through flow

330

cytometry. No significant differences between groups (i.e. with or without seminal plasma)

331

were observed in any of the evaluated sperm parameters.

15

332

3.3. Experiment 3

333

Pregnancy rates observed in the group of mares inseminated with epididymal frozen-

334

thawed sperm plus seminal plasma were significantly higher than in the control without

335

seminal plasma (16/25, 64% vs. 4/21, 19%; P<0.05).

336 337

4. Discussion

338

The present study investigates the preservation of epididymal stallion sperm in both

339

liquid and cryopreserved states. In the first experiment, we successfully maintained the

340

quality of stallion epididymal spermatozoa at 4ºC for up to 96 h. In effect, sperm motility and

341

viability were well maintained in samples stored at 4ºC, with values ranging from 70 to 80%,

342

which is in agreement with the results obtained by Brogan et al. [24]. Thus, the quality of

343

epididymal stallion sperm can be maintained at 4ºC using commercial extenders intended for

344

ejaculated semen. From a practical angle, these are striking results since they demonstrate that

345

the application of this technology could allow conserving epididymal sperm from animals of a

346

high genetic value that, for many reasons, have underwent castration. Most of the studies

347

reporting successful preservation of epididymal stallion sperm at 4°C have been performed

348

using the whole gonad [3, 5] or the epididymides [5,6]. In this regard, the current study

349

demonstrates that epididymal stallion sperm may be preserved at 4°C using milk or milk-egg

350

based commercial extenders. Apart from the advantages of preserving epididymal sperm cells

351

rather than the whole gonad, this outcome is very relevant because harvesting of sperm from

352

epididymis may be the last chance to obtain sperm from deceased, castrated or injured

353

stallions. Guimaraes et al. [2] demonstrated, for the first time, that extended epididymal

354

stallion sperm could be preserved at 4°C for a 24-h period without much affecting the sperm

355

quality. In the current study, we have shown that Gent and Kenney extenders are suitable to

16

356

preserve epididymal stallion sperm at 4ºC for up to 96 hours without affecting the sperm

357

quality, thus guiding the way to a possible use for AI. As aforementioned, Brogan et al. [24]

358

obtained similar results using a milk-based extender containing egg yolk, but the addition of

359

D-penicillamine, an antioxidant, showed detrimental effects. For this reason, these authors

360

concluded that the negative effect of D-penicillamine could reflect differences in the

361

physiology of ejaculated and epididymal spermatozoa, since the latter are not exposed to

362

seminal plasma.

363

Previous studies have reported differences in sperm motility and viability at different

364

periods of storage depending on the extender used, the egg yolk-based extenders showing

365

lower [3, 19] or higher motility and viability than those milk-based [15, 25-28]. Therefore,

366

while the composition of the extender may have an impact on the length of the cooling step

367

prior to freezing [23], our results show that, when used to store epididymal spermatozoa,

368

Kenney and Gent extenders present similar abilities to maintain their function and survival. In

369

addition, our motility and viability data for epididymal sperm were similar to those reported

370

in the literature for ejaculated semen. Therefore, and matching with Tiplady et al. [7], our

371

findings reinforce the idea that both Kenney and Gent extenders are suitable for short-term

372

liquid preservation of epididymal stallion sperm.

373

The second experiment was devised to determine whether supplementing frozen-

374

thawed epididymal sperm with seminal plasma improves sperm quality. Our hypothesis was

375

that addition of seminal plasma at post-thaw could be beneficial for sperm physiology and

376

could make the AI with frozen-thawed epididymal stallion sperm much more efficient.

377

Indeed, while seminal plasma has been shown to be detrimental when included in

378

cryopreservation extenders for stallion semen [25, 29], its proteins are known to modulate the

379

uterine inflammatory reaction in different mammalian species (horses [12]; donkeys [20];

17

380

pigs [30, 31]; mice [32]). In addition, some seminal plasma proteins are decapacitation factors

381

that prevent premature capacitation of spermatozoa [33] and may modulate acrosome

382

reaction, as has been observed in bull [34] and boar spermatozoa [35]. There is great

383

variability and differing results along the literature about the effects of seminal plasma on

384

post-thaw motility of epididymal sperm. Thus, while some authors reported a negative effect

385

[34], others showed either beneficial [21], in that case depending on the seminal plasma dose

386

[14], or no-effects [3]. Our data indicate that the addition of seminal plasma to frozen-thawed

387

epididymal stallion sperm cause neither beneficial nor harmful effects on the quality of

388

epididymal stallion sperm. With regard to the use of freezing media for preserving epididymal

389

sperm, one should note that a previous study analyzed different cryoprotectants and

390

concluded that dimethylformamide is suitable for cryopreservation of equine epididymal

391

sperm, even yielding better results than glycerol [13]. However, this previous study used high

392

glycerol concentrations (5% or 2.5% combined with dimethilformamide), which are higher

393

than those of commercial extenders. Other studies showed that low concentrations of glycerol,

394

either combined or without methylformamide, exhibit good quality of post-thaw epididymal

395

sperm, which matches with our results using INRA Freeze (2.5% glycerol) [15, 28].

396

Since, in Experiment 2, incubation of frozen-thawed epididymal stallion sperm had no

397

impact on their function and survival, we devised a third study aiming to address whether

398

infusion of seminal plasma after AI with post-thaw epididymal sperm could improve

399

reproductive performance. It is known that pregnancy rates may be improved when seminal

400

plasma is added to frozen-thawed ejaculated sperm, since seminal plasma modulates the

401

inflammatory response and prevents the sperm phagocytosis performed by uterine neutrophils

402

[20, 26, 35]. In our third experiment, we demonstrated that addition of seminal plasma to

403

frozen-thawed epididymal stallion sperm after AI increases pregnancy rates, which could be

18

404

explained by the role that this fluid plays in the communication between sperm and the uterus.

405

This hypothesis would match with previous studies using ejaculated semen [1], and suggests

406

that, in this scenario, the main role of seminal plasma is not related to its impact on sperm

407

physiology but rather on the female reproductive tract.

408

In conclusion, milk-based or milk-based with egg yolk extenders are suitable to store

409

stallion epididymal sperm at 4ºC for up to 96 hours. On the other hand, not only does the

410

addition of seminal plasma to frozen-thawed epididymal sperm have no impact on sperm

411

function and survival, but it does increase pregnancy rates when infused into the uterine body

412

after AI. This suggests that rather than exerting a direct impact on sperm physiology, its

413

effects are exerted via modulating the interactions between spermatozoa and the genital tract

414

of the mare.

415

19

416

Acknowledgments

417

The authors acknowledge the support from the Ministry of Science, Innovation and

418

Universities, Spain (Grants: RYC-2014-15581), and the Regional Government of Catalonia,

419

Spain (2017-SGR-1229).

420 421

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422

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plasma on fertility of fresh and frozen-thawed stallion epididymal spermatozoa. Anim

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Reprod Sci 118: 48-53.

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[2] Guimaraes T, Lopes G, Ferreira P, Leal I, Rocha A, 2012: Characteristics of stallion

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[3] Bruemmer JE, Reger H, Zibinski EL, Squires EL, 2002: Effect of storage at 5ºC on the

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motility and cryopreservation of stallion epididymal spermatozoa. Theriogenology 58: 405-

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[4] James AN, Green H, Hoffman S, Landry AM, Paccamonti D, Godke RA, 2002:

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[6] Stawicki RJ, McDonnell SM, Giguère S, Turner RM, 2016: Preganancy outcomes using

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[8] Papa FO, Melo CM, Fioratti EG, Dell’aqua JA, Zahn FS, Alvarenga MA, 2008: Freezing

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posthaw motility of equine epididymal spermatozoa. Theriogenology 28: 773-782.

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[10] Morris L, Tiplady C, Allen WR, 2002: The in vivo fertility of cauda epididymal

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spermatozoa in the horse. Theriogenology 58: 643-646.

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[11] Cary JA, Madill S, Farnsworth K, Hayna JT, Duoos L, Fahning ML, 2004: A

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comparison of electroejaculation and epididymal sperm collection techniques in stallions.

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Can Vet J 45: 35-41.

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[12] Neild D, Miragaya M, Chaves G, Pinto M, Alonso A, Gambarotta M, Losinno L,

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Aquero A, 2006: Cryopreservation of cauda epididymis spermatozoa from slaughterhouse

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testicles 24 h after ground transportation. Anim Reprod Sci 94: 92-95.

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[13] Olaciregui M, Gil L, Montón A, Luño V, Jerez RA, Martí JI, 2014: Cryopreservation of

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epididymal stallion sperm. Cryobiology 68: 91-95.

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[14] Neuhauser S, Dörfel S, Handler J, 2014: Dose-dependent effects of homologous seminal

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plasma on motility and kinematic characteristics of post-thaw stallion epididymal

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spermatozoa. Andrology 3: 536-543.

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[15] Neuhauser S, Bollwein H, Siuda M, Handler J, 2019: Comparison of the effects of five

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semen extenders on the quality of frozen-thawed equine epididymal sperm. Journal of Equine

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Veterinary Science 79: 1-8.

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[16] Barker CA, Gandier SCC, 1957: Pregnancy in a mare resulting from frozen epididymal

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spermatozoa. Can J Comp Med 21: 47-51.

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[17] Bruemmer JE, 2006: Collection and freezing of epididymal stallion sperm. Vet Clin

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North Am: Eq Pract 22: 677.

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[18] Alghamdi AS, Foster DN, Troedsson MH, 2004: Equine seminal plasma reduces sperm

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binding to polymorphonuclear neutrophils (PMNs) and improves the fertility of fresh semen

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inseminated into inflamed uteri. Reproduction 127: 593–600.[6]

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[19] Palm F, Walter I, Budik S, Aurich C, 2006: Influence of different semen extenders and

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seminal plasma on the inflammatory response of the endometrium in oestrous mares. Anim

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Reprod Sci 94: 286–289.

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[20] Miró J, Vilés K, García W, Jordana J, Yeste M, 2013: Effect of donkey seminal plasma

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on sperm movement and sperm-polymorphonuclear neutrophils attachment in vitro. Anim

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Reprod Sci 140:164-72.

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[21] Stout TAE, Morris LHA, Li X, Allen WR, 1999: The effect of seminal plasma on the

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motility and cryopreservability of horse epididymal sperm. In: Havemeyer Foundation

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[22] Boye JK, Katzman SA, Kass PH, Dujovne GA, 2019: Effects of lidocaine on ejaculated

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sperm and epididymal sperm post-castration. Theriogenology 134: 83-89.

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[23] Yeste M, Estrada E, Rocha LG, Marin H, Rodríguez-Gil JE, Miró J, 2015:

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Cryotolerance of stallion spermatozoa is related to ROS production and mitochondrial

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membrane potential rather than to the integrity of sperm nucleus. Andrology 3: 395-407.

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[24] Brogan PT, Beitsma M, Henning H, Gadella BM, Stout TAE, 2015: Liquid storage of

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equine semen: Assessing the effect of D-penicillamine on longevity of ejaculated and

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epididymal stallion sperm. Animal Reproduction Science 159: 155-162.

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[25] Rota, A, Furzi, C, Panzani, D, Camillo F, 2004: Studies on motility and fertility of

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cooled stallion spermatozoa. Reproduction in Domestic Animals 39: 103-109.

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of

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plasma

on

cooled-preserved

Amiata

donkey

spermatozoa.

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[27] Heitland AV, Jasko DJ, Squires EL, Graham JK, Pickett BW, Hamilton C, 1996: Factors

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affecting motion characteristics of frozen-thawed stallion spermatozoa. Equine Veterinary

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Journal 28: 47-53.

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[27] Rigby SL, Brinsko SP, Cochran M, Blanchard TL, Love CC, Varner DD, 2001:

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Advances in cooled semen technologies: seminal plasma and semen extender. Anim Reprod

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Sci 68: 171-180.

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[28] Melo CM, Papa FO, Fioratti EG, Villaverde AISB, Avanzi BR, Monteiro G, Dell’aqua

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JA, Pasquini DF, Alvarenga MA, 2008. Comparison of three different extenders for freezing

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epididymal stallion sperm. Anim Reprod Sci 107: 331

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[29] Barrier-Battut I, Bonnet C, Giraudo A, Dubois C, Caillaud M, Vidament M, 2013:

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Removal of seminal plasma enhances membrane stability on fresh and cooled stallion

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spermatozoa. Reprod Domest Anim 48:64-71.

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[30] Taylor U, Rath D, Zerbe H, Schuberth HJ, 2008: Interaction of Intact Porcine

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Spermatozoa with epithelial cells and neutrophilic granulocytes during uterine passage.

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Reprod Domest Anim 43:166–175.

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[31] Li JC, Yamaguchi S, Funahashi H, 2012: Boar seminal plasma or hen’s egg yolk

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decrease the in vitro chemotactic and phagocytotic activities of neutrophils when co-

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incubated with boar or bull sperm. Theriogenology 77:73–80.

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[32] Robertson SA, Mau VJ, Tremellen KP, Seamark RF, 1996: Role of high molecular

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weight seminal vesicle proteins in eliciting the uterine inflammatory response to semen in

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mice.J Reprod Fertil 107:265–277.

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[33] Robertson SA, 2005: Seminal plasma and male factor signaling in the female

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reproductive tract. Cell Tissue Res 322: 43–52.

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[34] Therien I, Soubeyrand S, Manjunath P, 1997: Major proteins of bovine seminal plasma

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modulate sperm capacitation by high-density lipoprotein. Biol Reprod 57: 1080-1088.10]

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[35] Harkema W, Colenbrander B, Engel B, Woelders H, 2004: Effects of exposure of

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epididymal boar spermatozoa to seminal plasma on the binding of zona pellucida proteins

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during in vitro capacitation. Theriogenology 61: 215.

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24

522

Table 1. Sperm motility descriptors obtained by CASA. Parameter

Units

Description Measures

Curvilinear velocity (VCL)

µm/s progression

the along

sequential the

true

trajectory Measures the straight trajectory of the spermatozoa per unit Straight-line velocity (VSL)

µm/s time

Average pathway velocity (VAP)

µm/s Measures the mean trajectory of the spermatozoa per unit time

Linearity coefficient (LIN)

%

VSL/VCL × 100

Straightness coefficient (STR)

%

VSL/VAP × 100

Wobble coefficient (WOB)

%

VAP/VCL × 100 Measures the mean head displacement along the curvilinear

Lateral head displacement (mean ALH) µm/s trajectory The frequency with which the sperm trajectory crosses the Frequency of head displacement (BCF) Hz average path trajectory

523 524

25

525

Table 2. Percentages of viability, total motility and progressive motility of epididymal

526

spermatozoa stored at 4ºC for 96 h using two different extenders (Kenney and Gent), and after

527

freeze-thawing (FT). Kenney

Gent

Time Viability (%)

TMOT (%)

PMOT (%)

Viability (%)

TMOT (%)

PMOT (%)

0h

86.6±3.1a

72.3±4.1a

65.5±10.1a

81.70±4.9a

70.5±5.3a

65.4±12.0a

24 h

83.8±3.6a

71.4±2.3a

66.3±8.9a

84.11±3.8a

72.6±6.2a

66.3±9.6a

48 h

77.7±4.3a

69.7±4.0a

63.2±9.9a

69.73±11.2a

65±10.3a

62.8±14.5a

72 h

81.3±2.0a

71.2±3.8a

60.8±11.2a

67.47±12.4a

62.8±12.3a

60.3±14.5a

96 h

76.9±4.3a

71.3±8.1a

63.2±12.3a

76.90±1.9a

65.8±14.0a

61.8±13.5a

FT

65.2±2.5b

56.5±6.5b

32.6±13.8b

59.3±4.5b

53.4±6.8b

31.8±9.8b

528

Data are shown mean ± SEM. Different superscripts (a, b) mean significant (P<0.05) differences between rows

529

within columns. There were no significant differences between extenders. TMOT: Total motile spermatozoa;

530

PMOT: Progressively motile spermatozoa; FT: frozen-thawed.

531

532

Table 3. Sperm kinematic parameters of epididymal stallion sperm flushed by Kenney or Gent extenders after 0, 24, 48, 72 or 96 hours of

533

storage at 4ºC. Treatment

VCL

VSL

VAP

LIN

STR

WOB

ALH

BCF

G0h

60.26±2.70 26.68±1.54 39.66±2.03 40±1.00 65±1.00 60±1.00 2.31±0.09 7.03±0.22

K0h

65.47±2.51 31.79±1.54 43.81±1.88 44±1.00 70±1.00 62±1.00 2.47±0.08 7.47±0.18

G24h

43.65±2.47 23.68±1.66 31.22±2.03 45±1.40 69±1.20 63±1.00 1.65±0.07 6.73±0.20

K24h

47.41±2.06 24.15±1.34 31.02±1.50 43±1.00 69±1.00 60±08

G48h

69.19±3.18 42.13±2.39 63.47±2.75 55±1.80 74±1.50 71±1.30 2.26±0.10 8.03±0.26

K48h

55.07±2.14 30.64±1.65 38.01±1.77 46±1.20 71±1.00 62±1.00 2.11±0.07 7.18±0.17

G72h

81.51±3.74 52.07±2.99 65.84±3.30 60±2.00 77±2.00 76±1.00 2.49±0.01 8.87±0.29

K72h

33.63±2.04 18.24±1.51 21.92±1.61 48±2.00 76±1.00 61±1.00 1.39±0.07 7.20±0.27

G96h

98.01±4.48 46.94±2.79 67.59±3.48 49±3.00 72±3.00 67±2.00 3.36±0.17 11.40±0.56

K96h

64.42±5.26 34.65±3.77 43.90±3.43 51±3.00 78±3.00 63±3.00 2.44±0.18 8.87±0.65

1.64±0.07 6.72±0.19

534

Values are expressed as means ± SEM. (K) Kenney skin-milk extender; (G) Gent egg-yolk extender; (VCL) Curvilinear velocity (µm/s); (VSL) Straight-line velocity (µm/s);

535

(VAP) Average path velocityy (µm/s); (LIN) Linear coefficient (%); (STR) Straightness coefficient (%); (WOB) Wooble coefficient (%); (ALH) Lateral head displacement

536

(µm/sg); (BCF) Frequency of head displacement (Hz). No significant differences were observed between treatments (P>0.05).

537

538

Table 4. Sperm kinematic parameters of the four motile subpopulations identified in stallion epididymal sperm flushed by Kenney extender. Subpopulation

VAP

VCL

VSL

LIN

STR

WOB

ALH

BCF

1

8.98±1.05

18.60±1.46

5.40±0.91

2

39.27±1.63 68.99±2.28

3

84.80±1.61 107.86±2.25 73.64±1.41 69.49±1.71 87.45±1.83 79.35±1.60 3.29±0.11 10.05±0.39

4

96.50±2.35 150.23±3.27 28.56±2.06 19.45±2.49 31.20±2.67 65.00±2.33 5.47±0.16 10.01±0.57

29.82±1.11 58.04±1.19 49.91±1.04 1.02±0.07 4.78±0.25

27.90±1.44 43.65±1.73 73.03±1.86 59.10±1.62 2.94±0.11 8.10±0.40

539

Values are expressed as means ± SEM. (VCL) Curvilinear velocity (µm/s); (VSL) Straight-line velocity (µm/s); (VAP) Average path velocityy (µm/s); (LIN) Linear

540

coefficient (%); (STR) Straightness coefficient (%); (WOB) Wooble coefficient (%); (ALH) Lateral head displacement (µm/sg); (BCF) Frequency of head displacement (Hz).

541

No significant differences were observed between treatments (P>0.05).

542

543

Figure legends

544 545

Figure 1. Diagram of Experiment 1. After collection, epididymal sperm were diluted in

546

extender A (Kenney) or B (Gent) and stored at 4ºC (refrigerated) for 96 h, and cryopreserved

547

(frozen). This design was followed for 10 epididymal sperm samples collected from separate

548

stallions.

549 550

Figure 2. Percentages of sperm subpopulations after at 0, 24, 48, 72 and 96 h of storage in

551

Kenney extender at 4ºC (cooling). Values are expressed as percentages (%). Different

552

superscript letters mean significant differences (P<0.05) between storage times within a given

553

sperm subpopulation.

554 555

Figure 3. Effects of adding seminal plasma on motility (A-B) and functional parameters (C)

556

of epididymal frozen-thawed sperm. Motility parameters were analyzed by CASA. Sperm

557

viability was evaluated after SYBR14/PI staining, membrane lipid disorder was determined

558

with M540/YO-PRO-1, and intracellular Ca2+ levels were assessed with Fluo3/PI staining.

559

Percentages of spermatozoa with high levels of peroxides and superoxides were evaluated

560

through H2DCFDA/PI and HE/YO-PRO-1 tests, respectively.

ETHICAL STATEMENT

The ethical commission explained us that for our study was not necessary an ethical protocol. No live animals were used in our work. Semen was collected from epididymides of 10 mature stallions aged 2-7 years after animal castration or euthanasia. The animals came from the Veterinary Faculty Hospital, Autonomous University of Barcelona (Bellaterra, Cerdanyola del Vallès, Spain). On the other hand, seminal plasma was a pool derived from 3 stallions housed in the Equine Reproduction Service Veterinary Faculty, Autonomous University of Barcelona. The seminal plasma was obtained from an aliquot of raw semen obtained routinely from these stallions in order to produce seminal doses.

CONFLICT OF INTERESTS

There is no any Conflict of Interests related to this manuscript,“New insights into preservation of stallion epididymal sperm and effects of seminal plasma”.