Accepted Manuscript The Effect of Superoxide Dismutase Mimetic and Catalase on the Quality of PostThawed Goat Semen Mojtaba Shafiei, Mohsen Forouzanfar, Sayyed Morteza Hosseini, Mohammad Hossein Nasr Esfahani PII:
S0093-691X(15)00033-3
DOI:
10.1016/j.theriogenology.2015.01.018
Reference:
THE 13070
To appear in:
Theriogenology
Received Date: 24 July 2014 Revised Date:
7 January 2015
Accepted Date: 17 January 2015
Please cite this article as: Shafiei M, Forouzanfar M, Hosseini SM, Nasr Esfahani MH, The Effect of Superoxide Dismutase Mimetic and Catalase on the Quality of Post-Thawed Goat Semen, Theriogenology (2015), doi: 10.1016/j.theriogenology.2015.01.018. 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 Effect of Superoxide Dismutase Mimetic and Catalase on the Quality of Post-Thawed
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Goat Semen
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Mojtaba Shafiei a, Mohsen Forouzanfar a, Sayyed Morteza Hosseini b, Mohammad Hossein Nasr
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Esfahani c *
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a-Department of Biochemistry, Fars Science and Research Branch, Islamic Azad University, Fars, Iran.
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b - Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive
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Biomedicine, ACECR, Tehran, Iran
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c - Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center, Royan Institute
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for Biotechnology, ACECR, Isfahan, Iran.
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*Correspondence:
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Mohammad H. Nasr-Esfahani. Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center,
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Tel: + 98 3195015680, Fax: + 9831 95015687, Email:
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Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
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Abstract:
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MnTE is a cell permeable superoxide dismutase mimetic agent which can convert superoxide to
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hydrogen peroxide (H2O2). Supplementation of MnTE to commercial semen extender can
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protect sperm from superoxide but not H2O2. Therefore, we proposed that addition of catalase
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(0.0, 200 or 400 IU/ ml) in combination with MnTE (0.1 µM) may further improve the
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cryopreservation efficiency of goat semen in a commercially optimized freezing media like,
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Andromed®. Therefore, ejaculates were obtained from three adult bucks twice a week during
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breeding season and diluted with Andromed® supplemented with or without MnTE and catalase,
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and were frozen in liquid nitrogen. Sperm parameters and reactive oxygen species (ROS)
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contents were evaluated 2 hours after dilution (prefreezing) and following freezing/thawing. The
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results revealed that all the treatments ,significantly (P ≤0.05) improved sperm motility, viability,
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membrane integrity post freezing and reduced ROS content compared to control but maximum
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improvement was obtained in MnTE+ 400 IU/ml catalase. In addition, supplementation with these
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antioxidants significantly (P ≤0.05) increases the cleavage rate after in vitro fertilization. In conclusion,
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the results of present study suggest that addition of antioxidant MnTE or catalase to a
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commercial optimized media, like Andromed®, improves total motility, membrane integrity and
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viability of goat semen samples post thawing. But the degree of improvement for these
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parameters significantly (P ≤0.05) higher when MnTE and catalase were simultaneously added
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to the cryopreservation media.
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Keywords: Antioxidant, MnTE, Semen Cryopreservation, Sperm Motility, Reactive
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Oxygen Species.
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1. Introduction
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Despite advances in semen cryopreservation, a variety of injuries occur at cellular and molecular
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levels during semen cryopreservation which may impair sperm function and fertilization
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potential [1, 2]. Major drawbacks of semen cryopreservation are reduction of sperm viability,
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motility, plasma membrane integrity and impaired function of the survived cell population [3].
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These phenomena are accounted by osmotic stress, cold shock, intracellular ice crystal and
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oxidative stress induced by reactive oxygen species (ROS) [4]. Among the aforementioned
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factors, excessive production of ROS plays a central role in induction of cryoinjury. ROS acts as
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a double-edged sword in many physiological phenomena. Basal levels of ROS are required for
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normal sperm motility, capacitation and acrosome reaction while excess ROS lead to various
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forms of cellular damages, including impairment of membrane, DNA damage and apoptosis [5,
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6]. Somatic cells have acquired a delicate balance between ROS generation and ROS scavengers
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but unlike most somatic cells, sperm as one of the smallest cells of the body has little cytoplasm.
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This makes sperm susceptible to ROS injuries which can affect sperm motility [7], viability [8],
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DNA integrity and energy metabolism [9]. Moreover, ROS can also be produced by exogenous
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sources such as semen handling during cryopreservation process, exposure to light, oxygen and
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shearing forces generated by centrifugation. Therefore, supplementation of semen diluents with
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antioxidants has been advised and a wide variety of antioxidants have been used for semen
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cryopreservation in bovine [10], water buffalo [11], stallion [12], ram [13] and goat [14].
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Antioxidants used during semen cryopreservation can be divided into two main categories: cell
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permeable and impermeable antioxidants. In addition, antioxidants can be categorized as
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enzymatic, enzyme-mimetic and non-enzymatic. Recent researches have suggested that cell
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permeable enzyme-mimetic antioxidants are considered as valuable compounds for reducing
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intracellular ROS levels [15]. In this regard, we and others researchers have shown that addition
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of cell permeable enzyme-mimetic antioxidants to commercially optimized cryopreservation
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media with antioxidant activity can further improve the quality of these media [13, 14]. MnTE
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[Manganese (III) meso-tetrakis (N-ethylpyridinium-2-yl) porphyrin chloride] is a manganese
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porphyrin compound with a superoxide dismutase (SOD) mimetic activity which has been shown
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to successfully rescue animal models for some oxidative stress-related diseases [16]. We
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previously proposed that 0.1µM of MnTE can improve sperm membrane and acrosomal
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integrity. Furthermore, we showed that in vitro rate of blastocyst formation following freeze–
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thawing of goat semen supplemented with MnTE was higher compared to its corresponding
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control [14].
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SOD is one of the enzymatic antioxidants which converts superoxide anion (O2.-) into hydrogen
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peroxide (H2O2) and oxygen (O2), while catalase can convert this H2O2 into the oxygen and
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water [17, 18]. Therefore, supplementation with MnTE is expected to increase H2O2, which is
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relatively stable cell permeable molecule with high oxidant potential. In other words the
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association of SOD mimetic (MnTE) and peroxide scavengers (catalase) may promote better
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sperm freezing performance. Therefore, the aim of this study was to address whether addition of
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catalase in combination with MnTE or alone, into commercially optimized freezing media could
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further improve the efficiency of cryopreservation of goat semen.
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2. Materials and methods
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2.1. Chemicals
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All chemicals used in this study were purchased from Sigma Chemical CO. (St. Louis, MO,
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USA) unless stated otherwise. MnTE was a gift from Dr Ines Batinić-Haberle (Department of
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Radiation Oncology, Duke University Medical School, Durham, NC 27710, USA). Andromed®
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as semen extender was obtained from Minitube, Germany.
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2.2. Semen collection, processing and freezing-thawing
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Semen collection and processing procedures were carried out according to Forouzanfar et al.[14].
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In brief, ejaculates were obtained by artificial vagina from three adult Bakhtiari bucks aged 2-3
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years, twice a week during the breeding season (September /October, 2013) in the animal farm of
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Reproductive Biotechnology research center at Royan Institute (Isfahan, Iran latitude 32°39'N).
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Samples from each male were kept separated and transported at 35 °C to the laboratory for
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microscopic assessment within 30 min. Only ejaculates showing higher than 70% motile and
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80% morphologically normal sperm and more than 1 ml in volume were used for experiments.
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To minimize individual differences, semen samples from the three bucks in each replicate were
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pooled. A total of 6 ejaculates from each goat were processed. For freezing, all of the treatments were
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repeated six times with the pooled semen samples. Andromed® was used as the base freezing
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extender. Each pooled semen sample was divided into six equal aliquots and diluted (1:20 v/v)
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with extender containing no antioxidant (control), 0.1 µM of MnTE (Mn), 200 IU/ml catalase
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(CAT 200), 400 IU/ml catalase (CAT 400), 0.1 µM of MnTE plus 200 IU/ml catalase (Mn+CAT
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200), 0.1 µM of MnTE plus 400 IU/ml catalase (Mn+CAT 400). Concentration of MnTE and
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catalase were based on our previous report [14] and Roca et .al [19], respectively. The diluted
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semen was cooled to 4°C over a period of 2 hours and drawn into 0.5 ml French straws (Biovet,
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L’Agile France), heat-sealed and stored at 4°C one hour for more equilibration. The straws were
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exposed to liquid nitrogen (LN2) vapor for 12 min, plunged into LN2, and stored in LN2 until
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thawed and used for evaluation of sperm parameters and in vitro fertilization. Thawing was
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carried out by plunging sealed straws in a 37°C water bath for 30 seconds. The thawed samples
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were individually evaluated by a single trained individual.
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2.3. Semen evaluation after thawing 2.3.1. Measurement of sperm motility
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For assessment of sperm motility post thawing, four or five straws from one treatment in each
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group were thawed and pooled. The samples were diluted with fertilization medium (Tyrode's
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albumin lactate pyruvate medium- Fert-TALP) to final concentration of 1x106spz/ml. The
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percentages of rapid progressive (class A) , sperm that swim fast in a straight line, slow
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progressive (class B), sperm that move forward but tend to travel in a curved, and non-
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progressive (class C),sperm that do not move forward despite that they move their tails, as well
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as total motility which refers to the population of sperm that display any type of movement, were
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assessed according to the software setting of the computer-assisted sperm analysis (CASA)
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system (Video Test, ltd: version Sperm 2.1© 1990-2004, Russia) [20], that was set-up for goat
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sperm evaluation. For each sample, 5 µl of sperm suspension was loaded on a pre-warmed slide
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and then covered by a 18 x 18 mm coverslip, a minimum of 500 sperm per sample were analyzed
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in at least three different microscopic fields.
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2.3.2. Assessment of sperm plasma membrane integrity
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Sperm plasma membrane integrity was estimated by hypo-osmotic swelling test (HOST) based
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on curled and swollen tails [21]. For this purpose 25 µl of the diluted semen (for prefreezing or
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thawed specimen) were mixed with 200 µl of HOST solution (100 mOsm/l, 57.6 mM fructose
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and 19.2 mM sodium citrate) for 30 min at room temperature, subsequently 5 µl of homogenized
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mixture was mounted and directly investigated using an inverted microscope (Olympus, CKX41
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- Japan).The percentages of spermatozoa with swollen and curved tails, categorized as intact
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plasma membrane, were estimated from recording at least 200 spermatozoa in more than five
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different microscopic fields.
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2.3.3. Assessment of sperm viability
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Sperm viability was assessed using eosin-nigrosin staining which was done for prefreezing (2
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hours post semen dilution) and post thawing experiments with the same protocol. Fifty µl of
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diluted or thawed semen was mixed with 100 µl of eosin (1%) in a test tube for 30 second, the
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mixture then was stained with nigrosin (10%) for a further 30 second and mixed gently. One
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drop of stained semen was smeared on slide and allowed to air dry in a dust-free environment
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and examined directly at a magnification of 1000× under oil immersion using light microscope.
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Two hundred sperm were assessed for each replicates. Sperm head that were unstained were
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classified as “live sperm”, while sperm that appeared as pink or red were considered as “dead
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sperm”.
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We used flow cytometery (FAC Scan; Becton Dickinson, San Jose, CA) to determine of the ROS
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content in thawed and diluted semen as described previously [22].In brief, the samples from each
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treatment/replicate were centrifuged at 700 x g for10 min, the supernatant was discarded and the
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pellet was re-suspended in 1ml of phosphate buffer saline. The percentages of ROS-positive
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spermatozoa in each treatments were determined following incubating one million sperm per ml
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with 5µM of 2′, 7′-Dichlorofluorescin diacetate (DCF-DA) for 30 min at room temperature.
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Regarding mechanism of ROS reaction with DCF, it is important to note that once DCF-DA
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enters cells, it loses its ester group and upon interaction with ROS, it produces a fluorescence
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compound. Live sperm produce a detectable amount of physiological ROS and therefore, the
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sperm become DCF positive. In order to differentiate between the physiological amount of ROS
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and pathological ROS production, we reduced the concentration of DCF-DA and therefore,
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sperm producing super physiological concentration of DCF-DA were detectable at this 5µM
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concentration of DCF-DA.
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2.4. In vitro fertilization and embryo culture
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In vitro fertilization (IVF) and embryo culture were performed as described by Forouzanfar et al.
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[14]. Goat ovaries were recovered at the local slaughterhouse, placed in normal saline (0.9 %
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Sodium Chloride) at a temperature between 25°C and 35°C and then transported to the lab within
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2 hours. The cumulus-oocyte complexes (COCs) comprised of at least 4-10 layers of cumulus
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cells and oocytes with a uniform cytoplasm and homogenous distribution of lipid droplets in the
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cytoplasm were recovered by aspiration from follicles of more than 2 mm diameter on the
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surface of the ovaries and selected for the in vitro maturation (IVM). The selected COCs were
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washed three times in the aspiration medium (Hepes-Tissue Culture Media (H-TCM) + 10%
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Fetal Calf Serum (FCS) + 100 IU/ml heparin), and then cultured in maturation medium (TCM-
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199 + 10% FCS + 5 mg/ml follicle-stimulating hormone + 5 mg/ml luteinizing hormone + 0.1
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mM cysteamin )in 5% CO2 at 39 °C and maximum humidity for 20- 22 hours. For sperm
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preparation, five straws representing one freezing operation in each replicate were thawed,
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pooled and washed through two-gradient (40%–80% solutions) of Pure Sperm (Nidacon;
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Gothenburg, Sweden) to separate motile sperm with normal morphology by centrifugation (700
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g/ 15 min-RT). Matured COCs (totally 1254 for all treatments and control) were partially
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stripped of the cumulus cells and transferred into 100-µl drops of fertilization medium and
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fertilized in vitro at 39°C and 5% CO2, 5% O2 and 90% nitrogen atmosphere. IVF experiments
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were repeated three times using pooled frozen-thawed samples in each group. For IVF, sperm at
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final concentration of 2 × 105 sperm were co-incubated with 20-25 matured oocytes/100 µl
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droplet for 22 hours under the same gas atmosphere condition as for IVM. Presumptive zygotes
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were mechanically denuded of their cumulus cells via pipetting and cultured in modified
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Synthetic Oviduct Fluid (SOF) medium for the first 3 days post insemination (day 0 = day of
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insemination). Embryos were then transported to SOF medium supplemented with 10% Charcoal
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stripped serum and 1.5 mM glucose up to blastocyst stage. Subsequently cleavage, blastocyst and
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hatched rates in each group were assessed on days 3, 8 and 9 post insemination, respectively.
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2.5. Statistical analysis
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The results are reported as the mean ± standard error (SE) for each experiment. The
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analysis of variance (ANOVA) was used for treatments comparisons .One-way ANOVA with
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the Tukey multiple comparison post-hoc test was used to compare the different stimuli. P ≤ 0.05
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was considered statistically significant. For IVF treatments, Student’s t-test was used to compare
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mean, when the data from the supplemented groups (CAT 200, CAT 400 and Mn+CAT 400)
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were pooled and compared with control group. 3. Results
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Table 1 shows the effects of antioxidant(s) supplementation in freezing media on
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percentage of sperm motility parameters before freezing (2h after dilution) and after freeze-
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thawing. There were no significant (P ≤0.05) differences in total, and class A + B motility
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between treatments in pre-freezing conditions. Following thawing, CAT 400 and Mn+CAT 400
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groups resulted in better maintenance of total and class A+B motility compared to control and
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other treatments. Mn, CAT 200 and Mn+CAT 200 groups was better maintained total and class
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A+B motility after thawing compared to control. It is of interest to note that Mn+CAT 400
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treatment yield higher post-thaw motility compared to control and other treatments (p