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The ideal holding time for boar semen is 24 h at 17 °C prior to short-cryopreservation protocols
Accepted Manuscript The ideal holding time for boar semen is 24�h at 17�°C prior to shot-cryopreservation protocols Mariana A. Torres, Matheus S. Monteiro, Marina S. Passarelli, Frederico O. Papa, José Antônio Dell’Aqua, Jr., Marco Antônio Alvarenga, Simone M.M.K. Martins, André F.C. de Andrade PII:
S0011-2240(18)30265-7
DOI:
https://doi.org/10.1016/j.cryobiol.2018.12.004
Reference:
YCRYO 4036
To appear in:
Cryobiology
Received Date: 25 August 2018 Revised Date:
20 November 2018
Accepted Date: 13 December 2018
Please cite this article as: M.A. Torres, M.S. Monteiro, M.S. Passarelli, F.O. Papa, José.Antô. Dell’Aqua Jr., Marco.Antô. Alvarenga, S.M.M.K. Martins, André.F.C. de Andrade, The ideal holding time for boar semen is 24�h at 17�°C prior to shot-cryopreservation protocols, Cryobiology (2019), doi: https:// doi.org/10.1016/j.cryobiol.2018.12.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The ideal holding time for boar semen is 24 hours at 17ºC prior to shot-cryopreservation
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protocols
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3 Mariana A. Torresa, Matheus S. Monteiroa, Marina S. Passarellia; Frederico O. Papab, José
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Antônio Dell’Aqua Jrb, Marco Antônio Alvarengab; Simone M.M.K. Martinsa, André F.C. de
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Andradea
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Swine research center, School of Veterinary Medicine and Animal Science, University of São
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Paulo, Pirassununga – SP
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and Animal Science, São Paulo State University, Botucatu, São Paulo, Brazil
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Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine
*Correspondence: Prof. Dr. André F.C. de Andrade, Swine research center, School of Veterinary
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Medicine and Animal Science, University of São Paulo, Pirassununga – SP. Tel.: +55-19- 3565
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4017; E-mail: [email protected]
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ABSTRACT
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Boar semen cannot be immediately cryopreserved, it need be hold at 17 ºC prior to
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cryopreservation, holding time has been used to improve cryopreserved boar semen, since
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holding time allows a prolonged interaction between spermatozoa and seminal plasma
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components. However, until now only few periods of holding time have been studied, and boar
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semen had been held at 17 ºC for 24 hours to facilitate its manufacture. Thus, this experiment
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aims to study the effect several holding time (0, 4, 8, 12, 24, 28 and 32 hours) on boar
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spermatozoa post-thawed (PT) characteristics. Fifteen sperm-rich fractions of ejaculate were
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extended in Beltsville Thawing Solution and storage at 17 °C. After each holding time (0, 4, 8,
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12, 24, 28 and 32 hours), a sample was centrifuged, and sperm pellet was diluted in an extender
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composed of sugars, amino acids, buffers, 20% egg yolk (v/v), antibiotics, 2% glycerol as a
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cryoprotectant, and 2% methylformamide (v/v). Cryopreservation was performed with an
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automatic cryopreservation system. Cryopreserved boar semen was evaluated to spermatozoa
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kinetics, plasma and acrosomal membranes integrity, mitochondrial membrane potential,
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detection of superoxide anion, plasma membrane fluidity, and peroxidation. Twenty-four hours
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of holding increase total and progressive motility, rapid spermatozoa, and integrity of plasma and
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acrosome membranes. To mitochondrial membrane potential, thirty-two hours is needed.
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However, holding time was not able to control the superoxide anion amount neither membrane
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lipid peroxidation, and had no effects on membrane fluidity. Thus, to reach the best results of PT
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boar semen the ideal holding time is twenty-four hours.
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Keywords: Swine; spermatozoa; cryopreservation; cooling; membrane integrity
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1. Introduction
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The first boar semen cryopreservation, in pellets, dates from 1975 made by Pursel and Johnson
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[37]; however, the actual cryopreservation methods with “maxi” (5 mL) and “mini” straws have
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been used for almost 30 years [20]. Despite the use of cryopreserved boar semen not be recent, it
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is not used on large scale [55]. It is due to the damages on the plasma membrane and acrosome
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and reduces sperm motility [37, 42, 55], but also to the complexity in handling cryopreservation
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and thawing procedures [42]. The simplification of those procedures could increase its use [42].
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Short-cryopreservation protocol is routinely used to cryopreserve bull, ram and equine semen [1,
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12, 24] and have been recently used to cryopreserve boar spermatozoa [38,48,51-52]. However,
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boar spermatozoa seem to be more sensitive to the toxicity of glycerol on high temperatures
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compared to bull, ram and equine spermatozoa. Thus, the incorporation of holding time on this
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simple protocol could be an alternative to improve the post-thawed (PT) sperm quality
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parameters.
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Cryopreservation facilities are normally far from pig farm; thus, semen must be delivered for
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cryopreservation. For it, semen is diluted in a cooling extender (like BTS - Beltsville Thawing
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Solution) and hold at 15-17 ºC [50]. This period is generic named ‘holding time’, and besides
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facilitating boar semen cryopreservation, it is also described that increases spermatozoa
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cryotolerance [8, 15, 17, 50, 54]. Although it is known that boar spermatozoa need at least 3
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hours of holding time [54], and it is usually hold for 24 hours to facilitate its manufacture, the
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ideal holding time that ensure the best cryopreservation results remain unknown.
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Seminal plasma components participate in several physiologic pathways, such as the activation of
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spermatozoa motility, antioxidation, stabilization of plasma and acrosomal membranes,
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regulation of mitochondrial metabolism, protection against proteases inhibitors and cold-shock
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protection [21, 33, 34, 54]. However, the cervix holds the seminal plasma, preventing it from
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reaching far away female reproductive organs [17]. Holding time is described as responsible to
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increase spermatozoa cryotolerance [50, 53-54]. This effect is possible due the extended
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interaction between seminal plasma components and spermatozoa plasma membrane during
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holding time [26]. With its seminal plasma components could be attached on sperm plasma
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membrane (during holding) and be carried to distant parts of the female reproductive tract, and
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then exert their actions.
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It is well known that the use of holding time prior to cryopreservation improves PT boar
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spermatozoa quality. However, until now the studies in the literature had performed only assays
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with few holding time periods per experiment using long-cryopreservation protocol (i.e. 3, 10 and
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20 hours [14]; 0 and 24 hours [8]; 2 and 24 [16]; 2, 8 and 24 hours [50]; 3 and 24 hours [54]).
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Beyond the bounds, the present study brings to the literature a holding time study with seven
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evaluated times of holding boar semen at 17 ºC previous to short-cryopreservation protocol. So,
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as previous elucidated, this study aims to describe the effects of several holding time periods (0,
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4, 8, 12, 24, 28 and 32 hours) on cryopreserved boar spermatozoa. Specifically, the effects on
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spermatozoa kinetics, plasma and acrosomal membranes integrity, mitochondrial membrane
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potential, detection of superoxide anion, plasma membrane fluidity and peroxidation.
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2. Material and methods
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The Ethics Committee for the use of Animals of the School of Veterinary Medicine and Animal
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Science of the University of São Paulo had approved this experiment under protocol
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5664140316. All animal procedures performed according to the legal and ethical standards.
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2.1. Experimental design
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The experimental design is shown in Figure 1. The experiment was designed as a randomized
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block (each boar was considered a block), and the experimental units were 1/7 of boar ejaculate
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sperm-rich fraction. This study was designed to compare seven different times of hold boar
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semen at 17 ºC prior to cryopreservation. Samples for this assay were from five boars, each
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sample were split for the seven holding time, and cryopreserved individually in three trials. Two
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straws were used to evaluate the CASA parameters, and other two straws to flow cytometry
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analysis.
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The medium Botu-Sui® (composed of sugars, amino acids, buffers, 20% egg yolk [v/v],
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antibiotics, 2% glycerol as a cryoprotectant, and 2% methylformamide [v/v]) was developed and
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donated by Biotech-Botucatu- Ltda /ME (Botucatu, SP, Brazil). Beltsville Thawing Solution
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(BTS) was acquired from IMV Technologies (L'Aigle, France). The fluorescent probes Syto 59
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(S59), C11-BODIPY581/591 (BP), Yo-Pro-1 (YP), dihydroethidium (DHE), JC-1 and
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Merocyanine 540 (M540) were purchased from Molecular Probes (Eugene, OR). Unless
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otherwise stated, all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
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2.3. Incubation media
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Semen samples were diluted in Tyrode’s albumin lactate pyruvate (TALP) sperm medium [3].
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The medium pH was adjusted to 7.4 using 5 N NaOH. During staining, TALP medium was
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maintained at 37°C in a water bath.
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2.4. Semen collection, raw semen evaluation, and the simplified protocol to cryopreservation
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Three sperm-rich fraction (SRF) of ejaculate was obtained from each of five boars (n = 15) by
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the gloved-hand technique. Immediately after collection, each semen sample was held at room
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temperature (20 to 22 °C) in its own seminal plasma, without dilution, for 30 minutes [36, 44].
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Raw semen was evaluated to volume, spermatozoa concentration (Neubauer chamber),
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spermatozoa kinetics (computer-assisted sperm analysis – SCA, Microptics®- Barcelona/Spain)
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and morphology (differential interference contrast microscopy – DIC). After raw semen
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evaluation, it was extended 1:2 (v:v) in BTS, aliquoted in seven treatments (periods of holding
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time 0, 4, 8, 12, 24, 28 and 32 hours) and cooled at 17 °C. After each holding time, extended
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semen was centrifuged under 2200 x g for 3 minutes [7] and the supernatant was separated from
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the pellet by aspiration. Spermatozoa pellet was suspended with freezing extender to obtain a
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final concentration of 600 x 106 spermatozoa/mL [40] and stored in 0.5 mL straws (IMV
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Technologies, Laigle, France). Straws were subsequently cryopreserved with a short-
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cryopreservation protocol according Torres et al. [51-52] using an automatic freezing system (TK
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3000; TK Tecnologia em Congelação Ltda, Uberaba, Brazil) and cooled at a rate of −0.5°C/min
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to 5°C. The freezing rate used was −20°C/min from 5 to −120°C. Subsequently, the straws were
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immersed into liquid nitrogen at −196°C. All straws were kept in liquid nitrogen for a minimum
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of 1 wk before thawing.
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2.5. Semen thawing
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Two straws per ejaculate and time of holding were thawed in a water bath at 37 °C for 30 sec.
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thawed semen was directly diluted in BTS (1:2; v: v). Additionally, to thawed analysis diluted
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samples was extended another time to archive the ideal spermatozoa concentration for each
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sperm analysis. Computer-assisted semen analysis was performed after samples dilution in BTS
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to 50 x 106 spermatozoa/mL. Flow cytometer analysis was performed after semen dilution in
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TALP to 5 x 106 spermatozoa/mL.
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A sample aliquot (5 µL) was placed on a pre-warmed Makler chamber and evaluated by phase-
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contrast microscopy (Nikon, Model Eclipse 80i) with 100x magnification. Five good fields were
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examine using SCA (Microptics®- Barcelona/Spain) for evaluating the following parameters:
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total (TM) and progressive (PM) motility, rapid spermatozoa (RAPID), curvilinear velocity
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(VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), straightness
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(STR), amplitude of lateral head displacement (ALH), beat cross frequency (BCF) and
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hypermotility (HYPER). We evaluated the spermatozoa hyperactivated (ALH>3.5 µm and
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VCL>97 µm/s) using Edit/Sort in the software [45].
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2.7. Flow cytometry assays
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Flow cytometry analysis were performed with Accuri C6 flow cytometer (Becton, Dickinson and
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Company, San Jose, CA). All assays were performed according the modifications made by Díaz
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et al., [12]. Samples were staining with S59 (5 nM in 150 µL at 5 × 106 spermatozoa/mL; band
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pass filter of 675/25 nm) for 10 min at 37°C. S59 is a dye-permeable membrane cell to stain the
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DNA of the sperm cells. Therefore, particles with the same size and scatter properties as
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spermatozoa were not counted as spermatozoa [12, 51]. The spectral overlap of the staining in the
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flow cytometer analyses was compensated whenever necessary. The samples were processed
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through the instrument at an acquisition rate of approximately 600 to 1,000 events/s, acquiring
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10,000 S59 positive events per assay. The cells were simultaneously excited by an argon laser at
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488 nm and by a red laser at 640 nm.
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Plasma and acrossomal membranes were assessed with Propidium Iodide (PI; 3.3 µg/mL; band
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pass filter of 610/20 nm) and Pisum sativum agglutinin conjugated to fluorescein isothiocyanate
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(FITC-PSA; 0.6 mg/mL; band pass filter of 530/30 nm). Sperm samples were stained
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simultaneous with S59, PI and FITC-PSA [12], and incubated at 37°C for 10 minutes. After
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incubation the samples were diluted by the addition of 150 µL of TALP and were analyzed by
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flow cytometry [2,51].
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The functionality of electron transport chain was evaluated in live cells (PI negative) by 5,5’,6,6’-
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tetra-chloro-1,1’,3,3’-tetraethylbenzimidazolyl carbocyanine iodide (JC-1 – 1 µM; band pass
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filter of 585/40 nm). After dilution in TALP, PT samples were stained (37 °C/ 10 min) with S59,
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PI and JC-1 [12]. After incubation the samples were diluted by the addition of 150 µL of TALP
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and JC-1 mean fluorescence intensity of viable cells (PI negative) were evaluated by flow
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cytometry [12].
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The dihydroethidium (DHE; 2 µM; band pass filter of 585/40 nm) was used to assed (by median
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fluorescence intensity) the amount of O2- [41]. DHE was added to semen samples and incubated
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at 37 °C for 10 minutes. After incubation Yo-Pro1 (YP; 50 nM; band pass filter of 530/30 nm)
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was added and incubated for 10 minutes. Finally, cells were stained with S59 for 10 minutes [12].
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After incubation the samples were diluted by the addition of 150 µL of TALP and DHE median
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fluorescence intensity (arbitrary units) of viable cells (YP negative) were evaluated by flow
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cytometry.
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The fluidity of plasma membrane was assessed with Merocianine 540 (M540; 16.2µM; band pass
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filter of 585/40 nm). Samples were stained with YP, and after 10 min of incubation S59 was
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added and incubated for 10 minutes at 37 °C [12]. M540 was added followed by incubation for
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70 s [15 ,39]. After incubation the samples were diluted by the addition of 150 µL of TALP and
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M540 median fluorescence intensity (arbitrary units) of viable cells (YP negative) were evaluated
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by flow cytometry.
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C11-BODIPY581/591 probe (BP; 6.67 µg/mL; band pass of 530/30 nm) was used to evaluate
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peroxidation of spermatozoa membranes. Semen aliquots were stained with BP for 20 min at
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37°C [30]. After the incubation period, samples were stained with PI and S59 [12]. Ten minutes
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after, sperm were diluted in TALP to 2.5 × 106 spermatozoa/mL and were analyzed by flow
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cytometry. The green BP median intensity of fluorescence emission (arbitrary units) was
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analyzed by flow cytometry for the viable sperm (PI negative).
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196 2.8. Statistical analysis
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Holding time periods were considered the fixed variables, and boars were considered random
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variables. The residue normality and homogeneity of variances were verified, and when every
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necessary data was transformed by logarithm or arcsine function. Treatment effect were analyzed
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using the Tukey-Kramer SAS MIXED procedure (SAS® Studio, Copyright© 2012-2017, SAS
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Institute Inc., Cary, NC, USA). All statistical analyses were considered significant at p < 0.05,
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and all results are expressed as the means ± SEM.
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3. Results
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3.1. Computer-assisted sperm analysis (CASA)
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Cryopreservation of boar semen on the simplified protocol could be improved (p < 0.05) by the
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use of holding time from 4 hours, improving total, progressive motility and the percentage of
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rapid spermatozoa (Table 1). Furthermore, the highest (p < 0.05) percentage of progressive
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motility and rapid spermatozoa were obtained with 24 hours of HT compared to 0 hours of HT
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(Table 1). To total motility 8 and 24 hours of HT resulted on highest values compared to 0 hours 9
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of HT (p > 0.05; Table 1). All other spermatozoa characteristics evaluated by CASA (VCL, VSL,
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VAP, LIN, STR, ALH, BCF, HYPER) were not impaired (p > 0.05) by any time of holding
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(Table 1).
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Plasma and acrosomal membrane integrity and membrane fluidity were evaluated to verify the
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effects of HT. Spermatozoa cryopreservation after 4, 8, 24, 28 and 32 hours of holding are able to
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increase (p < 0.05) the percentage of spermatozoa with intact plasma and acrosomal membranes
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(IPIA; Figure 2). However, highest (p < 0.05) results are obtained after 24 hours of HT compared
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to 0 and 12 hours of HT (Figure 2). Furthermore, use of holding time does not change (p > 0.05)
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levels of plasma membrane fluidity (Figure 3A).
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Since holding time can affect sperm metabolism, mitochondrial membrane potential (∆ψm) of
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viable cells was analyzed. Cryopreservation of boar spermatozoa after 8 hours of incubation at 17
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°C increase (p < 0.05) ∆ψm of viable cells (Figure 3B). Furthermore, holding time of 32 hours
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result on the highest ∆ψm (p < 0.05) values compared to 0 and 4 hours of HT (Figure 3B).
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Oxidation effects of holding time were mensurated using superoxide anion levels and membrane
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lipid peroxidation. To both variables, holding time was deleterious. Lower oxidative changes
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were detected only at 0 hours (p < 0.05) of holding to detection of superoxide anion (Figure 3C)
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compared to 4, 8, 12, 24, 28, and 32 hours of HT and at 0, 4 and 8 hours (p < 0.05) to lipid
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peroxidation compared to 32 hours of HT (Figure 3D).
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4. Discussion
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Holding time is widely used to cryopreserve boar semen, to facilitate its manufacture semen is
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hold at 17 ºC for 24 hours. However, the ideal period that semen must be in contact with its own 10
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seminal plasma, without harmful effects, is unknow. The literature brings us some holding time
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results, that were discussed below. Since any of these studies evaluated long holding time
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periods, them were not able to demonstrate the increasing of PT sperm characteristics among
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evaluated time until 24 (in general), and then was observed an overall decrease of PT boar
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spermatozoa quality at longer periods of holding.
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The effect of holding time to motile characteristics was previously described and remains
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controversial. Wasilewska and Fraser [53] described that holding time of 24 hours at 10 °C
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improves total and progressive motility when compared to 2 hours at 17 °C. Further, PT
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spermatozoa total motility did not show any improvement with 3 or 24 hours of holding prior to
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cryopreservation, but progressive motility was benefited when incubated for 24 hours [54]. On
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the other hand, no differences on sperm cells motility were observed on post-thawed semen after
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3 or 24 hours of holding [19]. In the same way, Tomás et al. [50] showed that cooling boar
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spermatozoa prior to cryopreservation for long times (24 hours) did not improve total and
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progressive motility compared to short periods (2 and 8 hours). On another view of holding time
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effect, Eriksson et al. [14] demonstrated that 20 hours of cooling prior to cryopreservation could
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be deleterious to sperm cells motility compared to 3 and 10 hours of holding. These results were
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the opposite of our finds, in which, we found the beneficial effect of 24 hours of holding time to
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total and progressive motility
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Ejaculated spermatozoa are able to absorb low molecular weight seminal plasma proteins [26-
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27]. Thus, incubation of spermatozoa prior to cryopreservation in its own, slightly extended,
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seminal plasma could allow spermatozoa to incorporate a greater amount of proteins. In this way,
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seminal calcitonin could be incorporate to boar spermatozoa during holding time. This protein
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was highly positively correlated (r = 0.8581, p < 0.05) with sperm cells motility [29]. Therefore,
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it can explain our finds, which the beneficial effect of 24 hours of holding time to total and
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progressive motility. Pipan et al. [34] described some correlations between seminal plasma ionic
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environment and sperm functionality. These authors find a positive correlation of selenium levels
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in seminal plasma with total and progressive motility (r = 0.674, r = 0.563 respectively, p <
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0.05). However, these authors also described a negative correlation of calcium concentration in
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seminal plasma (r = -0.436, p < 0.05) with progressive motility, as calcium is known to regulate
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sperm motility. This regulation is concentration-dependent, in which low concentration stimulate
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sperm motility and high concentration can inhibit motility [4]. Some seminal prostosomes are
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responsible for carrying calcium [31] and they are able to bind and transfer its contents to
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spermatozoa [5]. However, a long exposition to seminal prostosomes can possibly increase
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intracellular calcium, which can explain why long incubation time (28 and 32 hours) did not
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show any improvement on sperm cells motility.
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The use of holding time to cryopreservation of boar spermatozoa showed beneficial effects, when
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used for 24 hours, corroborating with other studies. Twenty-four hours of holding time at 10 °C is
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better than 2 hours at 17 °C to plasma membrane integrity to most evaluated animals, but the
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same results were not observed to acrosome integrity [53]. Post-thawed plasma membrane
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integrity can be improved by increasing from 3 to 24 hours of holding, but, acrosome integrity
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did not follow this same improvement [54]. Short times of holding (3 hours) did not have the
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same beneficial effect to plasma membrane integrity than little longer (10 and 20 hours) holding
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times [14].
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Those finds can be explained by the hypothesis that the bind of sperm proteins (BSP) also known
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as spermadhesin have the ability to bind to spermatozoa plasma membrane after ejaculation via
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choline phospholipids. BSP superfamily proteins can be classified by their ability of binding to
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heparin (HBP, which include AQN-1, AQN-3, AWN) or not (PSP-I, PSP-II) [35, 43]. The
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heparin-binding protein AQN-1 is related to plasma membrane integrity [6], while AWN-1 and
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AQN-3 are related to acrosome stabilization [13]. In rams, BSP1 and BSP5 are able to improve
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sperm viability and revert cold-shock-induced damage on sperm membranes [32]. Furthermore,
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boar spermatozoa cryopreserved after holding time of 24 hours increases phosphorylation levels
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of serine residues of HSP70 (heat-shock protein 70 kDa) compared to 3 hours of holding. Heat-
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shock protein is able to protect spermatozoa of cold-shock, resulting in the improvement of sperm
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membrane integrity and higher cryotolerance [54]. Thus, it is possible to understand how 24
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hours of holding increase post-thawing integrity of plasma and acrosomal membranes. However,
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effect of BSP proteins is time and concentration-dependent [25, 27]. After long exposition (up to
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8 hours) to BSP proteins induces phospholipid and cholesterol efflux from the spermatozoa outer
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membrane, which in turn, induce fragility of plasma membrane and its susceptibly to cold-shock
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[48, 49], which may explain the fact that long periods of holding time impair some characteristics
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of semen.
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Wasilewska and Fraser [53] described that 24 hours at 10 °C is better than 2 hours at 17 °C to
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mitochondrial membrane potential. Endorsing our results, in which the percentage of live
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spermatozoa with high mitochondrial membrane potential increases after 4 hours of holding. It
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could be explained by the fact that the holding time may be required to balance some enzymes
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between seminal plasma and spermatozoa. Glutathione (GSH) and alkaline phosphatase (ALP)
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may allow the availability of fructose as a source of energy to spermatozoa mitochondria.
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Reduced glutathione is involved in spermatozoa fructolysis [35]. Alkaline phosphatase acts in the
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synthesis and availability of fructose for spermatozoa [22]. Since mitochondrial oxidative
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phosphorylation (of fructose - OXPHOS) is the main source energy of boar spermatozoa [18],
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these events seem to be crucial. Possibly as consequence of that improvement of mitochondrial
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membrane potential we observed an increase on lipid peroxidation (from 12 hours of holding)
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and on superoxide anion levels (from 4 hours of holding). It could be due to the use of
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antioxidation enzymes for fructose availability, as previous mentioned; and also, to the electron
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transfer chain (ETC) of OXPHOS stimulates the ROS (reactive oxygen species) production [18,
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47]. Thus, it could cause an imbalance of antioxidation and pro-oxidation agents. Furthermore,
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Pipan et al. [34] described that iron and selenium had a positive correlation (r = 0.469, r = 0.467
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respectively, p < 0.05) with plasma membrane integrity. Stored boar sperm (during holding) is
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under oxidative conditions, since its metabolism is active [23]. Thus, to maintain equilibration
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between pro-oxidative and antioxidants, avoiding oxidative damages, iron is consumed by Fe-
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dependent enzymes (as catalase), and selenium is consumed to maintain levels of glutathione
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peroxidase. Therefore, concentration of these ions possibly decreases during cooling and holding
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time, impairing on decrease of antioxidants potential. However, our finds regard oxidation effects
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do not corroborate to Yeste et al. [54], which described, to long-cryopreservation protocol, that
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levels of intracellular peroxide did not change on post-thawing when spermatozoa were
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cryopreserved with 3 or 24 hours of holding. Indeed, these oxidation changes do not impair
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integrity either fluidity of sperm membranes. Thus, the deleterious effect of ROS levels
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increment was not observed in this study. Despite the increased levels of ROS and
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lipoperoxidation, these may still be physiologic for the sperm cells. Furthermore, the possible
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harmful effects of oxidation are surpassed by the beneficial effects on membranes integrity and
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mitochondrial metabolism.
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During and after ejaculation the HBP bind to sperm surface and act as decapacitant proteins
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protecting spermatozoa to premature capacitation [35, 43]. Another molecule associated to
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seminal decapacitant potential is cholesterol [10], which half of seminal cholesterol is associated
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to exosomes [11]. Piehl et al. [33] demonstrate that the exosomes and liposomes are able to block
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capacitation cholesterol-dependent and the increase plasma membrane fluidity. However, the
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interaction between spermatozoa and seminal HPB, exosomes and liposomes seem to not be
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beneficiated by increase of holding times, thus cooling of boar spermatozoa prior to short-
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cryopreservation protocol did not show any improvement to plasma membrane stability in this
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study. Nevertheless, to the long-cryopreservation protocol the use of holding time of 24 hours at
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10 °C prior to cryopreservation was able to decrease plasma membrane permeability compared to
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incubation of 2 hours at 17 °C [53]. Sperm plasma membrane permeability and lipid disorder
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increase on thawed boar spermatozoa cryopreserved after short (3 hours) holding time compared
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to 24 hours of incubation [54].
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Therefore, our results showed that hold boar semen for twenty-four hours prior to short-
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cryopreservation protocol increase total and progressive motility, rapid spermatozoa, integrity of
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plasma and acrosome membranes. To mitochondrial membrane potential thirty-two hours is
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needed to reach the highest values. However, was not able to control amount of superoxide anion
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neither membrane lipid peroxidation and had no effects on plasma membrane fluidity. Thus, to
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reach the best results of PT boar semen the ideal holding time prior to cryopreservation is twenty-
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four hours.
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The authors state that they have no conflict of interest.
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Author contributions
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M.A.T. conceived and designed the research study, wrote the paper, performed the experiments
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and analyzed the results; M.S.M performed and analyzed the experiments and collaborated with
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the writing; M.S.P. performed the experiments; S.M.M.K.M collaborated with results analysis;
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AFCA collaborated in the design, the results analysis and writing.
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Funding
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This work was supported by the São Paulo Research Foundation – FAPESP [grant numbers
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2015/17620-7, 2016/09441-8 and 2016/24690-4]
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TABLES AND FIGURES
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Table 1 Mean (± SEM) effects of holding time on simplified protocol to cryopreservation of boar spermatozoal on motility characteristics.
0
4
8
12
28
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TM (%)
8.45 ± 1.11b
12.05 ± 1.47a,b
16.47 ± 2.55a
15.37 ± 2.66a,b
20.72 ± 3.33a
13.71 ± 1.68a,b
13.62 ± 2.62a,b
PM (%)
5.22 ± 0.82b
7.48 ± 1.05a,b
10.53 ± 1.86a,b
9.76 ± 2.10a,b
13.17 ± 2.75a
8.48 ± 1.18a,b
8.60 ± 1.91a,b
Rapid (%)
4.42 ± 0.75b
5.89 ± 1.05a,b
8.79 ± 1.72a,b
8.71 ± 2.03a,b
11.18 ± 2.50a
6.94 ± 1.15a,b
6.36 ± 1.46a,b
VCL(µm/s)
51.98 ± 2.22
50.84 ± 1.92
51.28 ± 2.31
48.09 ± 2.04
51.64 ± 2.64
50.82 ± 1.88
49.98 ± 2.98
VSL(µm/s)
24.36 ± 2.33
24.85 ± 2.14
24.94 ± 2.25
22.41 ± 2.56
25.50 ± 2.68
26.14 ± 2.17
25.26 ± 2.70
VAP(µm/s)
31.99 ± 2.30
31.63 ± 1.87
32.57 ± 2.24
29.45 ± 2.33
32.98 ± 2.45
32.91 ± 2.02
32.09 ± 2.55
LIN (%)
46.00 ± 2.91
46.96 ± 2.93
47.44 ± 3.11
45.50 ± 3.75
48.53 ± 3.43
51.00 ± 3.03
50.80 ± 3.47
STR (%)
74.66 ± 2.09
74.96 ± 2.03
74.52 ± 2.51
73.50 ± 2.92
75.47 ± 2.70
78.44 ± 1.77
77.68 ± 2.39
ALH (µm)
2.74 ± 0.08
2.72 ± 0.08
2.66 ± 0.07
2.57 ± 0.07
2.70 ± 0.09
2.59 ± 0.08
2.63 ± 0.09
BCF (Hz)
5.86 ± 0.20
5.72 ± 0.26
5.73 ± 0.25
5.42 ± 0.27
5.80 ± 0.33
6.00 ± 0.24
5.93 ± 0.24
Hyper (%)
1.17 ± 0.13
1.57 ± 0.25
1.72 ± 0.21
1.43 ± 0.19
1.40 ± 0.25
1.19 ± 0.20
1.30 ± 0.35
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Holding time periods (hours)
TM – total motility; PM – progressive motility; VCL – curviliniar velocity; VSL – straight-line velocity; VAP – average path velocity; LIN – linearity; STR –
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straightness; ALH – amplitude of lateral head displacement; BCF – beat cross frequency; Hyper – hyperactived spermatozoa. Within a row, means without a common superscript differed (p < 0.05)
Fig. 1. Mean (± SEM) effects of holding time on simplified protocol to cryopreservation of boar spermatozoal on plasma and acrosomal membranes integrity. a-e
Within graphics, means without a common superscript differed (p < 0.05).
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Fig. 2. Mean (± SEM) effects of holding time on simplified protocol to cryopreservation of boar spermatozoal on oxidative damage and membrane fluidity. (A)
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Within graphics, means without a common superscript differed (p < 0.05)
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Plasma membrane fluidity; (B) mitochondrial membrane potential; (C) intracytoplasmatic oxidative stress; (D) lipoperoxidation of plasma membrane.
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ACCEPTED MANUSCRIPT Boar semen cannot be immediately cryopreserved, it need be hold at 17ºC: what is the ideal holding time?
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Mariana A. Torresa, Matheus S. Monteiroa, Marina S. Passarellia; Frederico O. Papab, José Antônio Dell’Aqua Jrb, Marco Antônio Alvarengab; Simone M.M.K. Martinsa,
HIGHLIGHTS
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André F.C. de Andradea
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Boar semen cannot be immediately cryopreserved;
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The ideal holding time for boar semen is 24 hours at 17ºC prior to
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Holding time allow a better interaction among seminal plasma molecules and spermatozoa;
Hold boar semen prior to cryopreservation increases sperm motility and
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cryopreservation;
membranes integrity;
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Holding time was not efficiently to control oxidative damages;
S0011-2240(18)30265-7
DOI:
https://doi.org/10.1016/j.cryobiol.2018.12.004
Reference:
YCRYO 4036
To appear in:
Cryobiology
Received Date: 25 August 2018 Revised Date:
20 November 2018
Accepted Date: 13 December 2018
Please cite this article as: M.A. Torres, M.S. Monteiro, M.S. Passarelli, F.O. Papa, José.Antô. Dell’Aqua Jr., Marco.Antô. Alvarenga, S.M.M.K. Martins, André.F.C. de Andrade, The ideal holding time for boar semen is 24�h at 17�°C prior to shot-cryopreservation protocols, Cryobiology (2019), doi: https:// doi.org/10.1016/j.cryobiol.2018.12.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The ideal holding time for boar semen is 24 hours at 17ºC prior to shot-cryopreservation
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protocols
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3 Mariana A. Torresa, Matheus S. Monteiroa, Marina S. Passarellia; Frederico O. Papab, José
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Antônio Dell’Aqua Jrb, Marco Antônio Alvarengab; Simone M.M.K. Martinsa, André F.C. de
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Andradea
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Swine research center, School of Veterinary Medicine and Animal Science, University of São
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Paulo, Pirassununga – SP
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and Animal Science, São Paulo State University, Botucatu, São Paulo, Brazil
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Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine
*Correspondence: Prof. Dr. André F.C. de Andrade, Swine research center, School of Veterinary
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Medicine and Animal Science, University of São Paulo, Pirassununga – SP. Tel.: +55-19- 3565
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4017; E-mail: [email protected]
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ABSTRACT
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Boar semen cannot be immediately cryopreserved, it need be hold at 17 ºC prior to
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cryopreservation, holding time has been used to improve cryopreserved boar semen, since
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holding time allows a prolonged interaction between spermatozoa and seminal plasma
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components. However, until now only few periods of holding time have been studied, and boar
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semen had been held at 17 ºC for 24 hours to facilitate its manufacture. Thus, this experiment
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aims to study the effect several holding time (0, 4, 8, 12, 24, 28 and 32 hours) on boar
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spermatozoa post-thawed (PT) characteristics. Fifteen sperm-rich fractions of ejaculate were
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extended in Beltsville Thawing Solution and storage at 17 °C. After each holding time (0, 4, 8,
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12, 24, 28 and 32 hours), a sample was centrifuged, and sperm pellet was diluted in an extender
35
composed of sugars, amino acids, buffers, 20% egg yolk (v/v), antibiotics, 2% glycerol as a
36
cryoprotectant, and 2% methylformamide (v/v). Cryopreservation was performed with an
37
automatic cryopreservation system. Cryopreserved boar semen was evaluated to spermatozoa
38
kinetics, plasma and acrosomal membranes integrity, mitochondrial membrane potential,
39
detection of superoxide anion, plasma membrane fluidity, and peroxidation. Twenty-four hours
40
of holding increase total and progressive motility, rapid spermatozoa, and integrity of plasma and
41
acrosome membranes. To mitochondrial membrane potential, thirty-two hours is needed.
42
However, holding time was not able to control the superoxide anion amount neither membrane
43
lipid peroxidation, and had no effects on membrane fluidity. Thus, to reach the best results of PT
44
boar semen the ideal holding time is twenty-four hours.
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Keywords: Swine; spermatozoa; cryopreservation; cooling; membrane integrity
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1. Introduction
48
The first boar semen cryopreservation, in pellets, dates from 1975 made by Pursel and Johnson
49
[37]; however, the actual cryopreservation methods with “maxi” (5 mL) and “mini” straws have
50
been used for almost 30 years [20]. Despite the use of cryopreserved boar semen not be recent, it
51
is not used on large scale [55]. It is due to the damages on the plasma membrane and acrosome
52
and reduces sperm motility [37, 42, 55], but also to the complexity in handling cryopreservation
53
and thawing procedures [42]. The simplification of those procedures could increase its use [42].
54
Short-cryopreservation protocol is routinely used to cryopreserve bull, ram and equine semen [1,
55
12, 24] and have been recently used to cryopreserve boar spermatozoa [38,48,51-52]. However,
56
boar spermatozoa seem to be more sensitive to the toxicity of glycerol on high temperatures
57
compared to bull, ram and equine spermatozoa. Thus, the incorporation of holding time on this
58
simple protocol could be an alternative to improve the post-thawed (PT) sperm quality
59
parameters.
60
Cryopreservation facilities are normally far from pig farm; thus, semen must be delivered for
61
cryopreservation. For it, semen is diluted in a cooling extender (like BTS - Beltsville Thawing
62
Solution) and hold at 15-17 ºC [50]. This period is generic named ‘holding time’, and besides
63
facilitating boar semen cryopreservation, it is also described that increases spermatozoa
64
cryotolerance [8, 15, 17, 50, 54]. Although it is known that boar spermatozoa need at least 3
65
hours of holding time [54], and it is usually hold for 24 hours to facilitate its manufacture, the
66
ideal holding time that ensure the best cryopreservation results remain unknown.
67
Seminal plasma components participate in several physiologic pathways, such as the activation of
68
spermatozoa motility, antioxidation, stabilization of plasma and acrosomal membranes,
69
regulation of mitochondrial metabolism, protection against proteases inhibitors and cold-shock
70
protection [21, 33, 34, 54]. However, the cervix holds the seminal plasma, preventing it from
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reaching far away female reproductive organs [17]. Holding time is described as responsible to
72
increase spermatozoa cryotolerance [50, 53-54]. This effect is possible due the extended
73
interaction between seminal plasma components and spermatozoa plasma membrane during
74
holding time [26]. With its seminal plasma components could be attached on sperm plasma
75
membrane (during holding) and be carried to distant parts of the female reproductive tract, and
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then exert their actions.
77
It is well known that the use of holding time prior to cryopreservation improves PT boar
78
spermatozoa quality. However, until now the studies in the literature had performed only assays
79
with few holding time periods per experiment using long-cryopreservation protocol (i.e. 3, 10 and
80
20 hours [14]; 0 and 24 hours [8]; 2 and 24 [16]; 2, 8 and 24 hours [50]; 3 and 24 hours [54]).
81
Beyond the bounds, the present study brings to the literature a holding time study with seven
82
evaluated times of holding boar semen at 17 ºC previous to short-cryopreservation protocol. So,
83
as previous elucidated, this study aims to describe the effects of several holding time periods (0,
84
4, 8, 12, 24, 28 and 32 hours) on cryopreserved boar spermatozoa. Specifically, the effects on
85
spermatozoa kinetics, plasma and acrosomal membranes integrity, mitochondrial membrane
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potential, detection of superoxide anion, plasma membrane fluidity and peroxidation.
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2. Material and methods
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The Ethics Committee for the use of Animals of the School of Veterinary Medicine and Animal
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Science of the University of São Paulo had approved this experiment under protocol
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5664140316. All animal procedures performed according to the legal and ethical standards.
92 93
2.1. Experimental design
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The experimental design is shown in Figure 1. The experiment was designed as a randomized
95
block (each boar was considered a block), and the experimental units were 1/7 of boar ejaculate
96
sperm-rich fraction. This study was designed to compare seven different times of hold boar
97
semen at 17 ºC prior to cryopreservation. Samples for this assay were from five boars, each
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sample were split for the seven holding time, and cryopreserved individually in three trials. Two
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straws were used to evaluate the CASA parameters, and other two straws to flow cytometry
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analysis.
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The medium Botu-Sui® (composed of sugars, amino acids, buffers, 20% egg yolk [v/v],
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antibiotics, 2% glycerol as a cryoprotectant, and 2% methylformamide [v/v]) was developed and
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donated by Biotech-Botucatu- Ltda /ME (Botucatu, SP, Brazil). Beltsville Thawing Solution
106
(BTS) was acquired from IMV Technologies (L'Aigle, France). The fluorescent probes Syto 59
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(S59), C11-BODIPY581/591 (BP), Yo-Pro-1 (YP), dihydroethidium (DHE), JC-1 and
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Merocyanine 540 (M540) were purchased from Molecular Probes (Eugene, OR). Unless
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otherwise stated, all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
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2.3. Incubation media
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Semen samples were diluted in Tyrode’s albumin lactate pyruvate (TALP) sperm medium [3].
113
The medium pH was adjusted to 7.4 using 5 N NaOH. During staining, TALP medium was
114
maintained at 37°C in a water bath.
115 116
2.4. Semen collection, raw semen evaluation, and the simplified protocol to cryopreservation
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boar semen 5
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Three sperm-rich fraction (SRF) of ejaculate was obtained from each of five boars (n = 15) by
119
the gloved-hand technique. Immediately after collection, each semen sample was held at room
120
temperature (20 to 22 °C) in its own seminal plasma, without dilution, for 30 minutes [36, 44].
121
Raw semen was evaluated to volume, spermatozoa concentration (Neubauer chamber),
122
spermatozoa kinetics (computer-assisted sperm analysis – SCA, Microptics®- Barcelona/Spain)
123
and morphology (differential interference contrast microscopy – DIC). After raw semen
124
evaluation, it was extended 1:2 (v:v) in BTS, aliquoted in seven treatments (periods of holding
125
time 0, 4, 8, 12, 24, 28 and 32 hours) and cooled at 17 °C. After each holding time, extended
126
semen was centrifuged under 2200 x g for 3 minutes [7] and the supernatant was separated from
127
the pellet by aspiration. Spermatozoa pellet was suspended with freezing extender to obtain a
128
final concentration of 600 x 106 spermatozoa/mL [40] and stored in 0.5 mL straws (IMV
129
Technologies, Laigle, France). Straws were subsequently cryopreserved with a short-
130
cryopreservation protocol according Torres et al. [51-52] using an automatic freezing system (TK
131
3000; TK Tecnologia em Congelação Ltda, Uberaba, Brazil) and cooled at a rate of −0.5°C/min
132
to 5°C. The freezing rate used was −20°C/min from 5 to −120°C. Subsequently, the straws were
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immersed into liquid nitrogen at −196°C. All straws were kept in liquid nitrogen for a minimum
134
of 1 wk before thawing.
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2.5. Semen thawing
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Two straws per ejaculate and time of holding were thawed in a water bath at 37 °C for 30 sec.
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thawed semen was directly diluted in BTS (1:2; v: v). Additionally, to thawed analysis diluted
139
samples was extended another time to archive the ideal spermatozoa concentration for each
140
sperm analysis. Computer-assisted semen analysis was performed after samples dilution in BTS
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to 50 x 106 spermatozoa/mL. Flow cytometer analysis was performed after semen dilution in
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TALP to 5 x 106 spermatozoa/mL.
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A sample aliquot (5 µL) was placed on a pre-warmed Makler chamber and evaluated by phase-
146
contrast microscopy (Nikon, Model Eclipse 80i) with 100x magnification. Five good fields were
147
examine using SCA (Microptics®- Barcelona/Spain) for evaluating the following parameters:
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total (TM) and progressive (PM) motility, rapid spermatozoa (RAPID), curvilinear velocity
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(VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), straightness
150
(STR), amplitude of lateral head displacement (ALH), beat cross frequency (BCF) and
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hypermotility (HYPER). We evaluated the spermatozoa hyperactivated (ALH>3.5 µm and
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VCL>97 µm/s) using Edit/Sort in the software [45].
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2.7. Flow cytometry assays
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Flow cytometry analysis were performed with Accuri C6 flow cytometer (Becton, Dickinson and
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Company, San Jose, CA). All assays were performed according the modifications made by Díaz
157
et al., [12]. Samples were staining with S59 (5 nM in 150 µL at 5 × 106 spermatozoa/mL; band
158
pass filter of 675/25 nm) for 10 min at 37°C. S59 is a dye-permeable membrane cell to stain the
159
DNA of the sperm cells. Therefore, particles with the same size and scatter properties as
160
spermatozoa were not counted as spermatozoa [12, 51]. The spectral overlap of the staining in the
161
flow cytometer analyses was compensated whenever necessary. The samples were processed
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through the instrument at an acquisition rate of approximately 600 to 1,000 events/s, acquiring
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10,000 S59 positive events per assay. The cells were simultaneously excited by an argon laser at
164
488 nm and by a red laser at 640 nm.
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Plasma and acrossomal membranes were assessed with Propidium Iodide (PI; 3.3 µg/mL; band
166
pass filter of 610/20 nm) and Pisum sativum agglutinin conjugated to fluorescein isothiocyanate
167
(FITC-PSA; 0.6 mg/mL; band pass filter of 530/30 nm). Sperm samples were stained
168
simultaneous with S59, PI and FITC-PSA [12], and incubated at 37°C for 10 minutes. After
169
incubation the samples were diluted by the addition of 150 µL of TALP and were analyzed by
170
flow cytometry [2,51].
171
The functionality of electron transport chain was evaluated in live cells (PI negative) by 5,5’,6,6’-
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tetra-chloro-1,1’,3,3’-tetraethylbenzimidazolyl carbocyanine iodide (JC-1 – 1 µM; band pass
173
filter of 585/40 nm). After dilution in TALP, PT samples were stained (37 °C/ 10 min) with S59,
174
PI and JC-1 [12]. After incubation the samples were diluted by the addition of 150 µL of TALP
175
and JC-1 mean fluorescence intensity of viable cells (PI negative) were evaluated by flow
176
cytometry [12].
177
The dihydroethidium (DHE; 2 µM; band pass filter of 585/40 nm) was used to assed (by median
178
fluorescence intensity) the amount of O2- [41]. DHE was added to semen samples and incubated
179
at 37 °C for 10 minutes. After incubation Yo-Pro1 (YP; 50 nM; band pass filter of 530/30 nm)
180
was added and incubated for 10 minutes. Finally, cells were stained with S59 for 10 minutes [12].
181
After incubation the samples were diluted by the addition of 150 µL of TALP and DHE median
182
fluorescence intensity (arbitrary units) of viable cells (YP negative) were evaluated by flow
183
cytometry.
184
The fluidity of plasma membrane was assessed with Merocianine 540 (M540; 16.2µM; band pass
185
filter of 585/40 nm). Samples were stained with YP, and after 10 min of incubation S59 was
186
added and incubated for 10 minutes at 37 °C [12]. M540 was added followed by incubation for
187
70 s [15 ,39]. After incubation the samples were diluted by the addition of 150 µL of TALP and
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M540 median fluorescence intensity (arbitrary units) of viable cells (YP negative) were evaluated
189
by flow cytometry.
190
C11-BODIPY581/591 probe (BP; 6.67 µg/mL; band pass of 530/30 nm) was used to evaluate
191
peroxidation of spermatozoa membranes. Semen aliquots were stained with BP for 20 min at
192
37°C [30]. After the incubation period, samples were stained with PI and S59 [12]. Ten minutes
193
after, sperm were diluted in TALP to 2.5 × 106 spermatozoa/mL and were analyzed by flow
194
cytometry. The green BP median intensity of fluorescence emission (arbitrary units) was
195
analyzed by flow cytometry for the viable sperm (PI negative).
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196 2.8. Statistical analysis
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Holding time periods were considered the fixed variables, and boars were considered random
199
variables. The residue normality and homogeneity of variances were verified, and when every
200
necessary data was transformed by logarithm or arcsine function. Treatment effect were analyzed
201
using the Tukey-Kramer SAS MIXED procedure (SAS® Studio, Copyright© 2012-2017, SAS
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Institute Inc., Cary, NC, USA). All statistical analyses were considered significant at p < 0.05,
203
and all results are expressed as the means ± SEM.
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3. Results
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3.1. Computer-assisted sperm analysis (CASA)
207
Cryopreservation of boar semen on the simplified protocol could be improved (p < 0.05) by the
208
use of holding time from 4 hours, improving total, progressive motility and the percentage of
209
rapid spermatozoa (Table 1). Furthermore, the highest (p < 0.05) percentage of progressive
210
motility and rapid spermatozoa were obtained with 24 hours of HT compared to 0 hours of HT
211
(Table 1). To total motility 8 and 24 hours of HT resulted on highest values compared to 0 hours 9
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of HT (p > 0.05; Table 1). All other spermatozoa characteristics evaluated by CASA (VCL, VSL,
213
VAP, LIN, STR, ALH, BCF, HYPER) were not impaired (p > 0.05) by any time of holding
214
(Table 1).
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Plasma and acrosomal membrane integrity and membrane fluidity were evaluated to verify the
218
effects of HT. Spermatozoa cryopreservation after 4, 8, 24, 28 and 32 hours of holding are able to
219
increase (p < 0.05) the percentage of spermatozoa with intact plasma and acrosomal membranes
220
(IPIA; Figure 2). However, highest (p < 0.05) results are obtained after 24 hours of HT compared
221
to 0 and 12 hours of HT (Figure 2). Furthermore, use of holding time does not change (p > 0.05)
222
levels of plasma membrane fluidity (Figure 3A).
223
Since holding time can affect sperm metabolism, mitochondrial membrane potential (∆ψm) of
224
viable cells was analyzed. Cryopreservation of boar spermatozoa after 8 hours of incubation at 17
225
°C increase (p < 0.05) ∆ψm of viable cells (Figure 3B). Furthermore, holding time of 32 hours
226
result on the highest ∆ψm (p < 0.05) values compared to 0 and 4 hours of HT (Figure 3B).
227
Oxidation effects of holding time were mensurated using superoxide anion levels and membrane
228
lipid peroxidation. To both variables, holding time was deleterious. Lower oxidative changes
229
were detected only at 0 hours (p < 0.05) of holding to detection of superoxide anion (Figure 3C)
230
compared to 4, 8, 12, 24, 28, and 32 hours of HT and at 0, 4 and 8 hours (p < 0.05) to lipid
231
peroxidation compared to 32 hours of HT (Figure 3D).
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4. Discussion
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Holding time is widely used to cryopreserve boar semen, to facilitate its manufacture semen is
235
hold at 17 ºC for 24 hours. However, the ideal period that semen must be in contact with its own 10
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seminal plasma, without harmful effects, is unknow. The literature brings us some holding time
237
results, that were discussed below. Since any of these studies evaluated long holding time
238
periods, them were not able to demonstrate the increasing of PT sperm characteristics among
239
evaluated time until 24 (in general), and then was observed an overall decrease of PT boar
240
spermatozoa quality at longer periods of holding.
241
The effect of holding time to motile characteristics was previously described and remains
242
controversial. Wasilewska and Fraser [53] described that holding time of 24 hours at 10 °C
243
improves total and progressive motility when compared to 2 hours at 17 °C. Further, PT
244
spermatozoa total motility did not show any improvement with 3 or 24 hours of holding prior to
245
cryopreservation, but progressive motility was benefited when incubated for 24 hours [54]. On
246
the other hand, no differences on sperm cells motility were observed on post-thawed semen after
247
3 or 24 hours of holding [19]. In the same way, Tomás et al. [50] showed that cooling boar
248
spermatozoa prior to cryopreservation for long times (24 hours) did not improve total and
249
progressive motility compared to short periods (2 and 8 hours). On another view of holding time
250
effect, Eriksson et al. [14] demonstrated that 20 hours of cooling prior to cryopreservation could
251
be deleterious to sperm cells motility compared to 3 and 10 hours of holding. These results were
252
the opposite of our finds, in which, we found the beneficial effect of 24 hours of holding time to
253
total and progressive motility
254
Ejaculated spermatozoa are able to absorb low molecular weight seminal plasma proteins [26-
255
27]. Thus, incubation of spermatozoa prior to cryopreservation in its own, slightly extended,
256
seminal plasma could allow spermatozoa to incorporate a greater amount of proteins. In this way,
257
seminal calcitonin could be incorporate to boar spermatozoa during holding time. This protein
258
was highly positively correlated (r = 0.8581, p < 0.05) with sperm cells motility [29]. Therefore,
259
it can explain our finds, which the beneficial effect of 24 hours of holding time to total and
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progressive motility. Pipan et al. [34] described some correlations between seminal plasma ionic
261
environment and sperm functionality. These authors find a positive correlation of selenium levels
262
in seminal plasma with total and progressive motility (r = 0.674, r = 0.563 respectively, p <
263
0.05). However, these authors also described a negative correlation of calcium concentration in
264
seminal plasma (r = -0.436, p < 0.05) with progressive motility, as calcium is known to regulate
265
sperm motility. This regulation is concentration-dependent, in which low concentration stimulate
266
sperm motility and high concentration can inhibit motility [4]. Some seminal prostosomes are
267
responsible for carrying calcium [31] and they are able to bind and transfer its contents to
268
spermatozoa [5]. However, a long exposition to seminal prostosomes can possibly increase
269
intracellular calcium, which can explain why long incubation time (28 and 32 hours) did not
270
show any improvement on sperm cells motility.
271
The use of holding time to cryopreservation of boar spermatozoa showed beneficial effects, when
272
used for 24 hours, corroborating with other studies. Twenty-four hours of holding time at 10 °C is
273
better than 2 hours at 17 °C to plasma membrane integrity to most evaluated animals, but the
274
same results were not observed to acrosome integrity [53]. Post-thawed plasma membrane
275
integrity can be improved by increasing from 3 to 24 hours of holding, but, acrosome integrity
276
did not follow this same improvement [54]. Short times of holding (3 hours) did not have the
277
same beneficial effect to plasma membrane integrity than little longer (10 and 20 hours) holding
278
times [14].
279
Those finds can be explained by the hypothesis that the bind of sperm proteins (BSP) also known
280
as spermadhesin have the ability to bind to spermatozoa plasma membrane after ejaculation via
281
choline phospholipids. BSP superfamily proteins can be classified by their ability of binding to
282
heparin (HBP, which include AQN-1, AQN-3, AWN) or not (PSP-I, PSP-II) [35, 43]. The
283
heparin-binding protein AQN-1 is related to plasma membrane integrity [6], while AWN-1 and
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AQN-3 are related to acrosome stabilization [13]. In rams, BSP1 and BSP5 are able to improve
285
sperm viability and revert cold-shock-induced damage on sperm membranes [32]. Furthermore,
286
boar spermatozoa cryopreserved after holding time of 24 hours increases phosphorylation levels
287
of serine residues of HSP70 (heat-shock protein 70 kDa) compared to 3 hours of holding. Heat-
288
shock protein is able to protect spermatozoa of cold-shock, resulting in the improvement of sperm
289
membrane integrity and higher cryotolerance [54]. Thus, it is possible to understand how 24
290
hours of holding increase post-thawing integrity of plasma and acrosomal membranes. However,
291
effect of BSP proteins is time and concentration-dependent [25, 27]. After long exposition (up to
292
8 hours) to BSP proteins induces phospholipid and cholesterol efflux from the spermatozoa outer
293
membrane, which in turn, induce fragility of plasma membrane and its susceptibly to cold-shock
294
[48, 49], which may explain the fact that long periods of holding time impair some characteristics
295
of semen.
296
Wasilewska and Fraser [53] described that 24 hours at 10 °C is better than 2 hours at 17 °C to
297
mitochondrial membrane potential. Endorsing our results, in which the percentage of live
298
spermatozoa with high mitochondrial membrane potential increases after 4 hours of holding. It
299
could be explained by the fact that the holding time may be required to balance some enzymes
300
between seminal plasma and spermatozoa. Glutathione (GSH) and alkaline phosphatase (ALP)
301
may allow the availability of fructose as a source of energy to spermatozoa mitochondria.
302
Reduced glutathione is involved in spermatozoa fructolysis [35]. Alkaline phosphatase acts in the
303
synthesis and availability of fructose for spermatozoa [22]. Since mitochondrial oxidative
304
phosphorylation (of fructose - OXPHOS) is the main source energy of boar spermatozoa [18],
305
these events seem to be crucial. Possibly as consequence of that improvement of mitochondrial
306
membrane potential we observed an increase on lipid peroxidation (from 12 hours of holding)
307
and on superoxide anion levels (from 4 hours of holding). It could be due to the use of
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antioxidation enzymes for fructose availability, as previous mentioned; and also, to the electron
309
transfer chain (ETC) of OXPHOS stimulates the ROS (reactive oxygen species) production [18,
310
47]. Thus, it could cause an imbalance of antioxidation and pro-oxidation agents. Furthermore,
311
Pipan et al. [34] described that iron and selenium had a positive correlation (r = 0.469, r = 0.467
312
respectively, p < 0.05) with plasma membrane integrity. Stored boar sperm (during holding) is
313
under oxidative conditions, since its metabolism is active [23]. Thus, to maintain equilibration
314
between pro-oxidative and antioxidants, avoiding oxidative damages, iron is consumed by Fe-
315
dependent enzymes (as catalase), and selenium is consumed to maintain levels of glutathione
316
peroxidase. Therefore, concentration of these ions possibly decreases during cooling and holding
317
time, impairing on decrease of antioxidants potential. However, our finds regard oxidation effects
318
do not corroborate to Yeste et al. [54], which described, to long-cryopreservation protocol, that
319
levels of intracellular peroxide did not change on post-thawing when spermatozoa were
320
cryopreserved with 3 or 24 hours of holding. Indeed, these oxidation changes do not impair
321
integrity either fluidity of sperm membranes. Thus, the deleterious effect of ROS levels
322
increment was not observed in this study. Despite the increased levels of ROS and
323
lipoperoxidation, these may still be physiologic for the sperm cells. Furthermore, the possible
324
harmful effects of oxidation are surpassed by the beneficial effects on membranes integrity and
325
mitochondrial metabolism.
326
During and after ejaculation the HBP bind to sperm surface and act as decapacitant proteins
327
protecting spermatozoa to premature capacitation [35, 43]. Another molecule associated to
328
seminal decapacitant potential is cholesterol [10], which half of seminal cholesterol is associated
329
to exosomes [11]. Piehl et al. [33] demonstrate that the exosomes and liposomes are able to block
330
capacitation cholesterol-dependent and the increase plasma membrane fluidity. However, the
331
interaction between spermatozoa and seminal HPB, exosomes and liposomes seem to not be
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beneficiated by increase of holding times, thus cooling of boar spermatozoa prior to short-
333
cryopreservation protocol did not show any improvement to plasma membrane stability in this
334
study. Nevertheless, to the long-cryopreservation protocol the use of holding time of 24 hours at
335
10 °C prior to cryopreservation was able to decrease plasma membrane permeability compared to
336
incubation of 2 hours at 17 °C [53]. Sperm plasma membrane permeability and lipid disorder
337
increase on thawed boar spermatozoa cryopreserved after short (3 hours) holding time compared
338
to 24 hours of incubation [54].
339
Therefore, our results showed that hold boar semen for twenty-four hours prior to short-
340
cryopreservation protocol increase total and progressive motility, rapid spermatozoa, integrity of
341
plasma and acrosome membranes. To mitochondrial membrane potential thirty-two hours is
342
needed to reach the highest values. However, was not able to control amount of superoxide anion
343
neither membrane lipid peroxidation and had no effects on plasma membrane fluidity. Thus, to
344
reach the best results of PT boar semen the ideal holding time prior to cryopreservation is twenty-
345
four hours.
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The authors state that they have no conflict of interest.
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Author contributions
351
M.A.T. conceived and designed the research study, wrote the paper, performed the experiments
352
and analyzed the results; M.S.M performed and analyzed the experiments and collaborated with
353
the writing; M.S.P. performed the experiments; S.M.M.K.M collaborated with results analysis;
354
AFCA collaborated in the design, the results analysis and writing.
355 15
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Funding
357
This work was supported by the São Paulo Research Foundation – FAPESP [grant numbers
358
2015/17620-7, 2016/09441-8 and 2016/24690-4]
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References
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[1] F.C. Almeida, S.V Silva, H.M. Souza, W.A. Gomes, J.A.C. Lima Filho, A.A. Wicke, A.M.
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TABLES AND FIGURES
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Table 1 Mean (± SEM) effects of holding time on simplified protocol to cryopreservation of boar spermatozoal on motility characteristics.
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TM (%)
8.45 ± 1.11b
12.05 ± 1.47a,b
16.47 ± 2.55a
15.37 ± 2.66a,b
20.72 ± 3.33a
13.71 ± 1.68a,b
13.62 ± 2.62a,b
PM (%)
5.22 ± 0.82b
7.48 ± 1.05a,b
10.53 ± 1.86a,b
9.76 ± 2.10a,b
13.17 ± 2.75a
8.48 ± 1.18a,b
8.60 ± 1.91a,b
Rapid (%)
4.42 ± 0.75b
5.89 ± 1.05a,b
8.79 ± 1.72a,b
8.71 ± 2.03a,b
11.18 ± 2.50a
6.94 ± 1.15a,b
6.36 ± 1.46a,b
VCL(µm/s)
51.98 ± 2.22
50.84 ± 1.92
51.28 ± 2.31
48.09 ± 2.04
51.64 ± 2.64
50.82 ± 1.88
49.98 ± 2.98
VSL(µm/s)
24.36 ± 2.33
24.85 ± 2.14
24.94 ± 2.25
22.41 ± 2.56
25.50 ± 2.68
26.14 ± 2.17
25.26 ± 2.70
VAP(µm/s)
31.99 ± 2.30
31.63 ± 1.87
32.57 ± 2.24
29.45 ± 2.33
32.98 ± 2.45
32.91 ± 2.02
32.09 ± 2.55
LIN (%)
46.00 ± 2.91
46.96 ± 2.93
47.44 ± 3.11
45.50 ± 3.75
48.53 ± 3.43
51.00 ± 3.03
50.80 ± 3.47
STR (%)
74.66 ± 2.09
74.96 ± 2.03
74.52 ± 2.51
73.50 ± 2.92
75.47 ± 2.70
78.44 ± 1.77
77.68 ± 2.39
ALH (µm)
2.74 ± 0.08
2.72 ± 0.08
2.66 ± 0.07
2.57 ± 0.07
2.70 ± 0.09
2.59 ± 0.08
2.63 ± 0.09
BCF (Hz)
5.86 ± 0.20
5.72 ± 0.26
5.73 ± 0.25
5.42 ± 0.27
5.80 ± 0.33
6.00 ± 0.24
5.93 ± 0.24
Hyper (%)
1.17 ± 0.13
1.57 ± 0.25
1.72 ± 0.21
1.43 ± 0.19
1.40 ± 0.25
1.19 ± 0.20
1.30 ± 0.35
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Holding time periods (hours)
TM – total motility; PM – progressive motility; VCL – curviliniar velocity; VSL – straight-line velocity; VAP – average path velocity; LIN – linearity; STR –
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straightness; ALH – amplitude of lateral head displacement; BCF – beat cross frequency; Hyper – hyperactived spermatozoa. Within a row, means without a common superscript differed (p < 0.05)
Fig. 1. Mean (± SEM) effects of holding time on simplified protocol to cryopreservation of boar spermatozoal on plasma and acrosomal membranes integrity. a-e
Within graphics, means without a common superscript differed (p < 0.05).
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Fig. 2. Mean (± SEM) effects of holding time on simplified protocol to cryopreservation of boar spermatozoal on oxidative damage and membrane fluidity. (A)
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Within graphics, means without a common superscript differed (p < 0.05)
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Plasma membrane fluidity; (B) mitochondrial membrane potential; (C) intracytoplasmatic oxidative stress; (D) lipoperoxidation of plasma membrane.
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ACCEPTED MANUSCRIPT Boar semen cannot be immediately cryopreserved, it need be hold at 17ºC: what is the ideal holding time?
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Mariana A. Torresa, Matheus S. Monteiroa, Marina S. Passarellia; Frederico O. Papab, José Antônio Dell’Aqua Jrb, Marco Antônio Alvarengab; Simone M.M.K. Martinsa,
HIGHLIGHTS
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André F.C. de Andradea
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Boar semen cannot be immediately cryopreserved;
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The ideal holding time for boar semen is 24 hours at 17ºC prior to
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Holding time allow a better interaction among seminal plasma molecules and spermatozoa;
Hold boar semen prior to cryopreservation increases sperm motility and
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cryopreservation;
membranes integrity;
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Holding time was not efficiently to control oxidative damages;