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Evaluation of ram semen enrichment with oleic acid on different spermatozoa parameters during low temperature liquid storage
Accepted Manuscript Title: Evaluation of ram semen enrichment with oleic acid on different spermatozoa parameters during low temperature liquid storage Author: Elham Zadeh Hashem Roya Haddad Mohsen Eslami PII: DOI: Reference:
S0921-4488(17)30060-3 http://dx.doi.org/doi:10.1016/j.smallrumres.2017.03.002 RUMIN 5440
To appear in:
Small Ruminant Research
Received date: Revised date: Accepted date:
22-9-2016 2-3-2017 3-3-2017
Please cite this article as: Hashem, E.Z., Haddad, R., Eslami, M.,Evaluation of ram semen enrichment with oleic acid on different spermatozoa parameters during low temperature liquid storage, Small Ruminant Research (2017), http://dx.doi.org/10.1016/j.smallrumres.2017.03.002 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.
Title: Evaluation of ram semen enrichment with oleic acid on different spermatozoa parameters
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during low temperature liquid storage
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Elham Zadeh Hashem1*, Roya Haddad2, Mohsen Eslami2
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Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
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Department of Theriogenology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
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Running Title: Ram semen enrichment with oleic acid
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*To whom correspondence should be addressed: Elham Zadeh Hashem, D.V.M., Ph.D.,
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Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
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Tel: +984432774737 ; Fax: +984432777099 ; E-mail: [email protected]
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ABSTRACT
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The purpose of the current experiment was to evaluate the protective effect of oleic acid on ram
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spermatozoa parameters during storage at refrigerator temperature. Ejaculates were collected
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from rams, pooled, diluted with extender and enriched with 0 (control), 0.125 (O 0.125), 0.25 (O
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0.25), 0.5 (O 0.5) and 1 (O1.0) mM oleic acid at a final concentration of 500×106
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spermatozoa/mL. Spermatozoa kinematics, viability and spermatozoa membrane integrity were
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evaluated by using computer-assisted sperm analysis (CASA), eosin-nigrosin staining and hypo-
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osmotic swelling (HOS) test, respectively. In addition, amounts of malondialdehyde (MDA),
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total antioxidant capacity (TAC), nitric oxide (NO) and superoxide dismutase (SOD) activities
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were assessed in the medium and spermatozoa at 0, 24, 48 and 72 h of storage. Enrichment of
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semen with oleic acid resulted in a significant (P < 0.05) increase in total motility, forward
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progressive motility and VCL (curvilinear velocity, µm/s) of O 0.5 and O1.0 groups at 48 and 72
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h of storage. In addition, viability and spermatozoa membrane functionality were greater in O 0.5
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(81.81 ± 2.29 % and 72.10 ± 2.63 %) and O1.0 (82.32 ± 2.36 % and 74.53 ± 1.90 %) groups
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compared to the control group (73.28 ± 1.60 % and 59.34 ± 2.12 %) at 72 h (P < 0.05). The
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measurement of MDA levels in spermatozoa showed that, there were higher in control group
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(0.84 ± 0.02 and 0.86 ± 0.04 µmol/g protein) in comparison with the O 0.125 (0.67 ± 0.05 and
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0.69 ± 0.03 µmol/g protein), O 0.25 (0.62 ± 0.03 and 0.66 ± 0.02 µmol/g protein), O 0.5 (0.62 ±
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0.02 and 0.65 ± 0.06 µmol/g protein) and O1.0 (0.60 ± 0.01 and 0.61 ± 0.01 µmol/g protein)
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groups at 48 and 72 h of storage (P = 0.002). Moreover, higher amounts of TAC were obtained
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in oleic acid treated groups compared to control group in spermatozoa and medium at 48 and 72
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h (P < 0.05). Nitrosative status was lower and SOD activities were higher in spermatozoa of oleic
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acid treated groups than the control group at 48 and 72 h (P < 0.05). Our results indicated that
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oleic acid with the dose of 0.5 and 1 mM would enhances ram semen quality during liquid
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storage at 48 and 72 h.
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Keywords: Oleic acid, Ram semen, Sheep, Total antioxidant capacity, Malondialdehyde.
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1. Introduction
One of the most important domestic species due to having triple productive profiles of milk,
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lamb and wool are sheep (Evans and Maxwell, 1987). Artificial insemination (AI), which is
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performed by using fresh or cooled diluted semen in sheep, makes the dissemination of genetic
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material from a small number of superior males to a large number of females possible (Maxwell
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and Watson, 1996). Storage of diluted semen has been widely used in AI programs. When the
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insemination is done within a short period of time after collection, diluted and cooled ram semen
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is a practical alternative to frozen semen to avoid freezing/thawing induced injuries (King et al.,
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2004). In small ruminants, the success of the AI technique depends on various factors especially
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those related with the semen processing and preservation.
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The temperature at which lipids change from the fluid to the crystalline state named phase
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transition temperature of plasma membrane (Foulks, 1977). Long time storage of spermatozoa
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above freezing point causes membrane deterioration, because of membrane phase transition
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occurring in the spermatozoa plasma membrane (Graham and Foot, 1987). One of the causes of
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decreased sperm viability following semen storage has been stated as the production of reactive
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oxygen species (ROS), which are generated from the metabolism of oxygen (O2) (Aitken et al.,
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1993). The mammalian spermatozoa is a redox active cell, which generates ROS spontaneously.
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Excessive ROS which is produced during liquid semen storage initiates oxidative reactions that
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eventually lead to the death of the sperm cells (De Lamirande and Gagnon, 1999; Guerra et al.,
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2004). To protect the spermatozoa against the harmful effects of the lipid peroxidation (LPO),
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several researches have been made to enrich semen by adding substances such as taurine, royal
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jelly, SOD, catalase, trehalose, glutathione, glutathione peroxidase, cytochrome c, glutamine and
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hyaluronan to ram semen (Maxwell and Stojanov, 1996; Bucak and Tekin, 2007; Bucak et al.,
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2009b; Moradi et al., 2013). Nevertheless, improvement in semen quality during cold storage
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still appears to be a challenge in sheep industry.
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Beneficial effects of oleic acid, the n-9 monounsaturated fatty acid with a 18-carbon chain, on
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total anti-oxidant capacity (TAC) and signal transduction alteration during in vitro (De Vries et
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al., 1997; Maedler et al., 2003; Menendez et al., 2006; Eslami et al., 2016; on rat ardiomyocytes,
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human pancreatic beta-cell, human breast, ovarian and stomach cancer cells and rooster sperm,
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respectively) and in vivo (Parthasarathy et al., 1990; Ruiz-Gutiérrez et al., 1999, on rabbit and
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rat, respectively) experiments have been well documented. However, the effect of oleic acid on
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ram semen during liquid storage has not yet been reported. Therefore, the current trial was
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performed to evaluate the effect of oleic acid on spermatozoa motion characteristics (evaluated
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by computer assisted sperm analysis; CASA), viability, spermatozoa plasma membrane
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functionality, amounts of malondialdehyde (MDA), TAC, nitric oxide (NO) and superoxide
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dismutase (SOD) activity of ram semen during low temperature liquid storage.
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2. Materials and Methods
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2.1. Animals and semen collection
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Animals were housed at the Research Farm of Agriculture Faculty of Urmia University,
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Urmia, Iran (Nazloo campus, East longitude 44◦, 58׳, 30 ״and North latitude 37◦, 39׳, 30 )״and
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maintained under uniform nutritional conditions (free access to good quality of hay and water).
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Ejaculates were collected from five mature Qezel rams (3 to 4 years of age; mean body weight=
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62.5 ± 3.1), twice a week with the aid of an artificial vagina in the presence of estrus ewes during
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the winter (four weeks; January 2016 to February 2016). Immediately after collection, semen
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was transferred to the laboratory and placed in a water bath (37 ˚C) for semen evaluation. The
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semen samples were evaluated for volume, sperm concentration, and percentage of motility.
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Only ejaculates between 0.75-2 mL volume with a concentration of greater than 2.5 × 109
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sperm/mL having >80% forward progressive motile sperm were selected and pooled for further
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analysis. Seven pooled ejaculates (35 individual semen samples; each ram gave seven samples)
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were used in the present experiment. The Animal Care committee of the Urmia University
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approved the semen sampling procedure.
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2.2. Semen processing and evaluation
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After pooling, semen was diluted using a tris-based extender containing of Tris
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(hydroxymethyl aminomethane) 3.63 g, fructose 0.5 g, citric acid 1.99 g, egg yolk 14% (v/v),
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100,000 IU penicillin and 100 mg streptomycin dissolved in distilled water (up to 100 mL), to a
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final concentration of 500×106 sperm/mL was used for medium of spermatozoa (Salamon and
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Maxwell, 2000). Each pooled semen sample was divided into 5 equal aliquots to treat with
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different concentrations of the oleic acid (1 ml for each treatment group at each time point). Both
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extender (medium) and semen were at 37◦C when mixed. Then, diluted semen was enriched with
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0 (control), 0.125 (O 0.125), 0.25 (O 0.25), 0.5 (O 0.5), and 1 (O1.0) mmolar bovine serum
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albumin (BSA)-conjugated oleic acid. Following enrichment, the semen was stored for 72 h at
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4◦C. The CASA (Test Sperm 2.1; Videotest, St. Petersburg, Russia) parameters (including total
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and forward progressive motility: FPM), percentage of viability and percentage of spermatozoa
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with healthy membrane were evaluated at 0, 24, 48 and 72 h of storage. Moreover, amounts of
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TAC, MDA, NO, activity of SOD and protein were measured in the medium and the
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spermatozoa at the mentioned time points. Oleic acid was purchased from Sigma Company (O
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1008, Sigma-Aldrich) and other materials used in this project were bought from the Merck
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Company.
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2.3. Preparation of BSA conjugated oleic acid
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Oleic acid was conjugated with BSA according to the method described by Van Harken et al.
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(1969) for the better utilization of oleic acid by spermatozoa. In brief, BSA solution (24%) was
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prepared in NaCl 0.09%. Then, 0.12g oleic acid was dissolved in 3 ml ethanol 95% and
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neutralized with NaOH. Then dissolved 0.13 g Na-oleate in 15 ml sodium chloride (0.9%). The
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Na-oleate was subsequently mixed with BSA solution and stored in the freezer -20 °C until used
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in the study.
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2.4. Spermatozoa kinematics evaluated by CASA
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A computer assisted sperm analyzer was used to assess spermatozoa kinematics. This system
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included a phase contrast microscope (Olympus, BX41, Tokyo, Japan) with a stage warmer, a
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CCD-camera (SDC-313B, Samsung Techwin Co., Gyeong, Korea). Videotaping of sperm
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motility was conducted at a rate of 25 frames/s. To assess spermatozoa kinematics, aliquot of
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extended semen was more diluted (25×106 cell/mL) with the same extender in each group. A 10
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µL of extended semen was placed on a warmed (37 ◦C) slide and covered with a cover slip
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(22mm×22 mm). The motion characteristics of spermatozoa was assessed at 37 ◦C at 200×.
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For each sample seven to eight fields per drop were analyzed and a minimum of 200
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spermatozoa were evaluated. The sperm variables included in the analysis, were total motility,
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FPM, curvilinear velocity (VCL, µm/s), straight-line velocity (VSL, µm/s), average path velocity
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(VAP,
µm/s),
linearity
(LIN
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[VSL/VAP]×100, %).
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2.5. Spermatozoa viability assessment
[VSL/VCL]×100,
%)
and
straightness
(STR
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The percentage of viability of spermatozoa in the control and oleic acid treated groups was
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assessed by means of eosin-nigrosin staining (Evans and Maxwell, 1987). The final composition
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of the stain was: eosin-Y 1.67 g, nigrosin 10 g, and sodium citrate 2.9 g, dissolved in distilled
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water (up to 100 mL). Spermatozoa suspension smears were prepared by mixing a 5µL of the
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semen sample with 10 µL of the stain on a warm slide and spreading the stain with a second
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slide. The viability was assessed by counting at least 200 cells under light microscope (1000×,
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oil immersion). Spermatozoa showing partial or complete purple color was considered non-
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viable and spermatozoa showing no color, indicating the complete exclusion of the stain were
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considered to be alive.
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2.6. Assessment of spermatozoa membrane functionality by using the hypo-osmotic swelling
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(HOS) test
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Spermatozoa membrane integrity was assessed by HOS test consisting of fructose (0.9% w/v)
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and sodium citrate solution (0.49% w/v, 100 mOsm/kg), which was prepared on a daily basis and
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kept at 4°C (Correa and Zavos, 1994). Diluted semen (20 µl) was added to the pre-warmed
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(37°C) HOS solution (200 µl) in a test tube and then incubated for 60 min at 37°C. Hypo-
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osmotic swelling test was undertaken under a phase contrast microscope (400×, Olympus, BX41,
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Tokyo, Japan), counting 200 sperms, to determine the percentage of spermatozoa with
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curled/swollen tail.
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2.7. Separation of spermatozoa from medium
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The spermatozoa and the medium were separated by centrifugation. Semen was centrifuged
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for 10 minutes at 550 g. The resulting pellet was used as the concentrated spermatozoa aliquot
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for purposes of the present study. The supernatant was centrifuged two more times, first for 10
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minutes at 550 g, and then for 30 minutes at 3000 g. The resulting supernatant was considered to
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be the medium for purposes of the present study (Blesbois et al., 1993). The pellet of
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spermatozoa was re-suspended in 1 ml phosphate buffer saline (PBS), then trichloroacetic acid
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50% was added to the PBS to break the spermatozoa membrane.
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2.8. Amounts of MDA in the spermatozoa and medium
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Amounts of MDA were measured using the thiobarbituric acid reaction, using the method
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described by Frederick (2010). Briefly, the spermatozoa suspension or medium was added to
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thiobarbituric solution (one volume sample to two volume solution), mixed, and then double
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distilled water was added and the mixture was shaken. Tubes were heated in boiling water, then
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cooled and centrifuged for 10 minutes at 1000 g. The absorbance of the upper layer was read at
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532 nm wave length. The amounts of MDA were expressed as µmol/g protein in the
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spermatozoa and medium samples.
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2.9. Amounts of TAC in spermatozoa and medium
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Amounts of TAC were measured using the methods of Koracevic et al. (2001) with a slight
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modification. In brief, 490 µl of PBS solution were added to 10 µl of the sample. Additionally,
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sodium benzoate, acetic acid, Fe-EDTA and H2O2 were added to the tubes, respectively. The
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tubes were incubated at 37 °C for 60 minutes. Acetic acid and thiobarbituric solution were
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subsequently added to the tubes. The tubes were incubated in 100 °C water bath for 10 minutes.
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The absorbance of the samples was measured at 532 nm wave length. Amounts of TAC were
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expressed as mmol/g protein in the spermatozoa and medium samples.
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2.10. Amounts of total NO in spermatozoa and medium The total NO values in the spermatozoa and medium were measured according to the Griess
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reaction (Green et al., 1982). In Griess reaction nitric oxide rapidly converted into the more
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stable nitrite, which in an acidic environment nitrite is converted to HNO2. In reaction with
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sulphanilamide, HNO2
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ethylenediamine·2HCl to form an azo dye that can be detected by absorbance at a wavelength of
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540 nm. The NO values were expressed as nmol/g protein in the spermatozoa and medium
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samples.
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2.11. Measuring the SOD activities in spermatozoa and medium
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forms a diazonium salt, which reacts with N-(1-naphthyl)
The SOD activities was evaluated by pyrogallol oxidation method. The total SOD activity
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was determined according to Marklund and Marklund (1974), assaying the auto oxidation and
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illumination of pyrogallol at 420 nm for 3.5 min. One unit total SOD activity was calculated as
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the amount of protein causing 50% inhibition of pyrogallol autooxidation. The total SOD activity
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was expressed as units per milligram of protein in the spermatozoa and medium samples.
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2.12. Total protein measurement in the spermatozoa and medium
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Total protein was measured according to Bradford protocol (1976). In brief, Bradford reagent
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and stock solution of bovine serum albumin were prepared. The Bradford reagent was
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subsequently added to the spermatozoa and medium samples. The absorbance of the contents of
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the test tubes was measured at 595 nm wave length after 15 minutes. Amounts of total protein
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were obtained using a standard curve developed for this purpose.
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2.13. Statistical analysis
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Values of spermatozoa kinematics, viability, spermatozoa membrane functionality and
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amounts of TAC, MDA, NO and SOD activities were expressed as the mean ± SEM.
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Percentage data were subjected to arcsine transformation. Means were analyzed using a one-way
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analysis of variance, followed by a Tukey’s post hoc test to determine significant differences in
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all the variables recorded among groups. Changes of the variables over time were analyzed using
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a Repeated Measure ANOVA to reveal the differences among different time points in each
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treatment group. All analyses were done using SigmaStat software (Version 3.5; Chicago, IL).
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For all statistical analyses, differences with P < 0.05 were considered significant.
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3. Results
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3.1. Spermatozoa motility variables assessed by CASA
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There were no significant differences in total motility among groups at 0 h and 24 h (P > 0.05;
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Table 1), while it was greater in O 0.25, O 0.5 and O1.0 groups compared to the control group at
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48 h of storage (Table 1; P = 0.004). Moreover, total motility was greater in oleic acid treated
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groups compared to control group at 72 h of storage (Table 1; P = 0.003). Within group analysis
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indicated that total motility were less at 72 h compared to 0 h in all treated groups (P < 0.023;
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Table 1). There were no significant differences in FPM among groups at 0 h (Table 1; P > 0.05).
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But, it was greater in O 0.5 and O1.0 groups compared to control groups at 24 h, 48 h and 72 h of
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storage (Table 1; P < 0.005). Moreover, FPM was greater in O 0.25 compared to control at 48 h,
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and O 0.125 and O 0.25 compared to control at 72 h of storage (Table 1; P < 0.002). Within
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group analyses revealed that the FPM was less at 24 h compared to 0 h, and 72 h compared to 24
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h in all groups (Table 1; P < 0.001). The VCL variable was significantly higher in oleic acid
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treated groups compared to control group at 72 h of storage (P < 0.001; Table 2). There were no
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significant differences between oleic acid treated groups and control group at other variables
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assessed by CASA (P > 0.05; Table 2).
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3.2. Viability of spermatozoa
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The interaction between time of storage and treatment indicated that the percent of viability
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did not differ among treatment groups at 0 h, 24 h and 48 h (P > 0.05; Table 3), but the percent
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of viability was greater in O 0.5 and O1.0 groups compared to control at 72 h of storage (P=
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0.04; Table 3). Within group analysis revealed that the percent of viability was less at 48 h and
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72 h compared to 0 h in all treated groups (P < 0.013; Table 3).
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3.3. Hypo-osmotic swelling test of spermatozoa
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The result of the present experiment revealed that oleic acid at proper concentration was able
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to protect plasma membrane of spermatozoa from damages during long time liquid storage (72h).
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There were no significant differences among groups in percent of spermatozoa with intact
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plasma membrane at 0 h and 24 h of storage (Table 4; P > 0.05). While, the percent of
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spermatozoa with intact plasma membrane was greater in O 0.25, O 0.5 and O1.0 groups
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compared to control group at 48 h and 72 h of storage (Table 4; P < 0.003). Changes over time
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indicated that the percent of spermatozoa with intact plasma membrane was lower at 72 h
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compared to 0 h in all treated groups (Table 4; P < 0.009).
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3.4. Malondialdehyde values (µmol/ g protein)
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3.4.1. Medium
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The interaction between time of storage and treatment revealed that the amounts of MDA did
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not differ among the treatment groups at 0 h and 24 h of storage (Fig. 1, P > 0.05), while MDA
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values were less in O 0.5 and O1.0 groups compared to control group at 48 h (Fig. 1, P = 0.041).
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Moreover, MDA values were greater in control group compared to O 0.125, O 0.25, O 0.5 and
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O1.0 groups at 72 h of storage (Fig. 1, P < 0.001). Within group analysis showed that the MDA
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values were increased during the experiment, and these increases were significant in all treated
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groups between 0 h and 48 h, and 72 h and 48 h of storage (Fig. 1, P < 0.001).
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3.4.2. Spermatozoa The results indicated that the MDA values (µmol/ g protein) of spermatozoa did not differ
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among the treatment groups at 0 h (Fig. 2, P > 0.05). Whereas, the amounts of MDA were less in
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O 0.5 and O1.0 groups compared to the control group at 24 h (Fig. 2, P = 0.016). Moreover, the
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values of MDA were significantly less in oleic acid treated groups compared to control group at
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48 h and 72 h of storage (Fig. 2, P = 0.002). Within group analysis revealed that amounts of
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MDA in spermatozoa were greater at 24 h, 48 h and 72 h compared to 0 h in control group (Fig.
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2, P = 0.002). Moreover, the MDA values were greater at 72 h compared to 0 h in O 0.5 group
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(Fig. 2, P = 0.046). There were no significant differences in O 0.125, O 0.25 and O1.0 groups
257
among different time points (Fig. 2, P > 0.05).
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3.5. Total TAC (mmol/ g protein)
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3.5.1. Medium
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Amounts of TAC were not differ among groups at 0 h and 24 h (Fig. 3, P > 0.05). While,
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amounts of TAC were greater in oleic acid treated groups compared to the control group at 48 h
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and 72 h of storage (Fig. 3, P = 0.003). Within group analysis revealed that TAC values were
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lower at 48 h and 72 h compared to 0 h in control, O 0.125, O 0.25 and O1.0 groups (Fig. 3, P <
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0.004), whereas, there were no significant differences among different time points in O 0.5 group
265
(P = 0.112; Fig. 3).
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3.5.2. Spermatozoa
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The interaction between time of storage and treatment indicated that significant differences
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were not observed for amounts of TAC among groups at 0 h (Fig. 4; P > 0.05). But, values of
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TAC were higher in O 0.5 (4.27 ± 0.16) and O1.0 (4.34 ± 0.13) groups compared to control (3.59
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± 0.18) group at 24 h of storage (Fig. 4; P = 0.036). Moreover, amounts of TAC were greater in
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O 0.25 (3.72 ± 0.10), O 0.5 (3.64 ± 0.15) and O1.0 (3.75 ± 0.25) groups compared to control
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(2.91 ± 0.16) at 48 h (Fig. 4; P = 0.030). On the other hand, the values of TAC were higher in
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oleic acid treated groups compared to control group at 72 h of storage (Fig. 4; P < 0.001). Within
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group analysis showed that the amounts of TAC in spermatozoa were less at 48 h and 72 h
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compared to 0 h in all treated groups (Fig. 4, P < 0.008).
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3.6. Total NO (nmol/g protein)
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3.6.1. Medium
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There were no significant differences in total amounts of NO among treatment groups at each
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time point (P > 0.05; Figure 5). Within group analysis showed that the levels of NO were greater
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at 48 h and 72 h compared to 0 h in all treated groups (P < 0.014; Fig. 5).
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3.6.2. Spermatozoa
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The interaction between time of storage and treatment showed that there were no significant
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differences for amounts of NO among groups at 0 h and 24 h, while amounts of NO were
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significantly less in oleic acid treated groups compared to the control group at 48 h and 72 h (P <
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0.011; Figure 6). Within group analysis indicated that the amounts of NO were significantly
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increased in a time dependency manner in the control group (Fig. 6; P < 0.001). Moreover,
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amounts of NO were greater at 72 h than the 0 h in O 0.5 and O1.0 groups (Fig. 6; P < 0.022).
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But, there were no significant differences among different time points in O 0.125 and O 0.25
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groups (Fig. 6; P > 0.05).
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3.7. Superoxide dismutase (unit/mg protein)
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3.7.1. Medium
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The measuring of SOD activities indicated that there were no significant differences among
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groups at each time point (P > 0.05; Figure 7). Within group analysis revealed that SOD
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activities were greater at 48 h than the 0 h in O 0.25 and O1.0 groups (Fig. 7; P < 0.037), but
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significant differences were not observed among different time points in other groups (Fig. 7; P >
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0.05).
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3.7.2. Spermatozoa
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The interaction between time of storage and treatment showed that there were no significant
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differences for SOD activities among groups at 0 h and 24 h (Fig. 8; P > 0.05), while SOD
300
activities were greater for oleic acid treated groups than the control group at 48 h and 72 h of
301
storage (Fig. 8; P < 0.011). Changes over time indicated that the activities of SOD were higher at
302
72 h compared to 0 h in O 0.125, O 0.25 and O 0.5 (Fig. 8; P < 0.039), while there were lower at
303
72 h compared to 0 h in control group (Fig. 8; P = 0.005).
an
us
cr
298
305
M
304
4. Discussion
Our experiment showed that oleic acid at the proper concentration and varying with time of
307
storage was able to promote total motility, forward progressive motility, VCL, percent of
308
viability, percent of spermatozoa with healthy plasma membrane, and amounts of total
309
antioxidant capacity and superoxide dismutase activities compared to control group. Moreover,
310
oleic acid ameliorates amounts of malondialdehyde and nitric oxide of ram spermatozoa during
311
low temperature liquid storage. To our knowledge, it the first report of oleic acid on different
312
spermatozoa parameters in small ruminants.
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306
313
In the present study, supplementation of ram semen with 0.5 and 1 mM oleic acid resulted in
314
14% and 16% increase in VCl at 24 h, 23% and 31% increase at 48 h and 29% and 40% increase
315
at 72 h compared to the control semen, respectively. On the other hand, enrichment of ram
316
semen with 0.5 and 1 mM oleic acid caused 16 % and 17% increase in FPM compared to the
14
Page 14 of 33
control semen at 24 h, 24% and 27% increase at 48 h and 29% and 30% increase at 72 h,
318
respectively. Then, oleic acid was able to improve the ram spermatozoa VCL at proper
319
concentration in according to time of storage. Moreover, adding of oleic acid to the ram semen at
320
the 0.5 and 1 mM caused more than 10% increase in viability at 72 h of storage. It has been
321
demonstrated that the fertilizing capacity of spermatozoa was correlated with quantitative
322
assessment of spermatozoa motility by CASA (Larsen et al., 2000). Moreover, among the
323
spermatozoa kinematics, VCL has been shown as the most significant and independent CASA
324
parameter, which highly correlates with the fertilization rate (Larsen et al., 2000). It seems that,
325
monounsaturated fatty acids could strengthen the antioxidant defense and PI3 kinase levels,
326
which are of the important factors in the viability of the cells (Oudit et al., 2004). Previous
327
studies indicated that semen supplementation with antioxidants resulted in higher viability and
328
motility of small ruminant spermatozoa [ram (Maxwell and Stojanov, 1996; Bucak et al., 2008),
329
goat (Bucak et al., 2010)]. However, bull semen enrichment with n-3 fatty acids and soft gels
330
containing n-3, n-6, n-9 fatty acids reduced the spermatozoa motility and viability of chilled and
331
frozen-thawed semen (Abavisani et al. 2013; Sheikholeslami Kandelousi et al. 2013). The
332
present study also indicated that spermatozoa viability and motility decreased during the
333
experiment. The probable reason for the reduction of spermatozoa viability and motility during
334
the cold storage period could be related to the endogenously produced ROS. Previous study
335
showed that the cooling process, which we provided in the experiments by storing the samples at
336
4 ◦C, stimulated the ROS production in spermatozoa (Sicherle et al., 2011).
Ac
ce p
te
d
M
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cr
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317
337
The amounts of polyunsaturated fatty acids in the spermatozoa membranes in small ruminant
338
is generally higher than in other species. Then, the spermatozoa of small ruminant is highly
339
susceptible to oxidative damage during storage, resulting from the production ROS (Gandini et
15
Page 15 of 33
al., 2000). Because of lower antioxidant capacity, spermatozoa could not counteract the
341
damaging effects of ROS and LPO during liquid storage. Then, the application of antioxidant to
342
the semen is mandatory to reduce the harmful effects of LPO during storage. It has been shown
343
that enrichment of goat semen with taurine reduced the MDA levels compared to the control
344
(Ateşşahin et al., 2008). On the other hand, adding of alpha lipoic acid to the buck (Ma et al.,
345
2011) and dog (Michael et al., 2007) semen resulted in lower LPO. While, supplementation of
346
ram semen with glutathione, oxidized glutathione or cysteine did not affect the MDA levels
347
(Bucak et al., 2008). The present study indicated that, oleic acid was able to reduce the MDA
348
levels in medium and ram spermatozoa during storage at refrigerator. It seems that the efficacy
349
of antioxidant on the amounts MDA would depends on concentration of antioxidant, type of
350
antioxidant, time of storage and animal species.
M
an
us
cr
ip t
340
The current experiment revealed that ram semen enrichment with oleic acid increased the
352
amounts of TAC in spermatozoa and medium. The results of the present work are compatible
353
with the previous reports about the improvement of TAC following supplementation of ram
354
semen with royal jelly, superoxide dismutase, taurine and cysteine (Marti et al., 2003; Bucak and
355
Tekin, 2007; Bucak et al., 2008; Moradi et al., 2013). Whereas, our result were inconsistent with
356
the findings of Bucak et al. (2009a) where it was reported that administration of antioxidants
357
glutamine and hyaluronan to the goat semen did not affect the TAC levels. Previous studies
358
showed that the increase in antioxidant indices following the treatment with monounsaturated
359
fatty acids could be a result of their potency to elevate essential enzymatic antioxidants (Narang
360
et al., 2004). It has been reported that SOD activity was found to be higher in the presence of
361
curcumin and carnitine compared to the semen of the control group in goat (Bucak et al., 2010).
362
Also, elevated SOD activity was observed following supplementation of bull semen with
Ac
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351
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Page 16 of 33
cysteine (Sarıözkan et al. 2009). While, other antioxidants (Glutamin and hyaloronan) treatment
364
decreased the SOD activity in goat and ram semen (Bucak et al., 2009 a,b). Our result verify the
365
findings of previous work about the ability of monounsaturated fatty acids in the elevation of
366
SOD activities in treated cell (Narang et al., 2004). The results of the present work indicated that,
367
with the prolongation of storage time, amounts of TAC were decreased and MDA and NO levels
368
were increased. It is suggesting the key role of the oxidative/nitrosative stress in long time liquid
369
storage period. The results of the present experiment are compatible with the previous reports
370
about the detrimental effects of long liquid storage on the levels of generated MDA and NO
371
(Gundogan et al., 2010; Moradi et al., 2013).
372
5. Conclusion
an
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363
In conclusion, our results showed the protective effects of the oleic acid supplementation on
374
ram sperm viability and plasma membrane integrity during liquid storage condition. The current
375
study also showed that oleic acid decreased the amounts of malondialdehyde and nitric oxide,
376
increased the total antioxidant capacity levels and superoxide dismutase activities and ultimately
377
enhanced the kinematics of ram spermatozoa compared to the control during low temperature
378
liquid storage. It seems that, the protective effects of oleic acid at 48h and 72h likely related to its
379
antioxidant property, which could be provided at suitable concentrations (0.5 and 1 mM).
380
Acknowledgments
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373
381
The present experiment was funded by the Research Deputy of the Urmia University. We are
382
grateful to Dr. Farhad Farrokhi-Ardabili for his kind assistance during the semen sampling
383
process.
384
References Abavisani, A., Arshami, J., Naserian, A.A., Sheikholeslami Kandelousi, M.A., Azizzadeh, M., 2013.
17
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Quality of bovine chilled or frozen-thawed semen after addition of omega-3 fatty acids supplementation to extender. Int. J. Fertil. Steril. 7, 161–168. Aitken, R.J., Harkiss, D., Buckingham, D., 1993. Relationship between iron catalyzed lipid peroxidation
ip t
potential and human sperm function. J. Reprod. Fertil. 98, 257–265. Ateşşahin, A., Bucak, M.N., Tuncer, P.B., Kizil, M. 2008. Effects of anti-oxidant additives on
cr
microscopic and oxidative parameters of Angora goat semen following the freeze-thawing process. Small Rumin. Res. 77, 38-44.
us
Blesbois, E., Grasseau, I., Blum, J.C., 1993. Effects of Vitamin E on fowl semen storage at 4◦C. Theriogenology 39, 771–779.
an
Bradford, M.M., 1976. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 7, 248-254.
M
Bucak, M.N. Ateşşahin, A., Yüce, A., 2008. Effect of anti-oxidants and oxidative stress parameters on ram semen after the freeze-thawing process. Small Rumin. Res. 75, 128-134.
d
Bucak, M.N., Sariözkan, S., Tuncer, P.B., Sakin, F., Ateşşahin, A., Kulaksiz, R., Ҫevik, M., 2010. The
te
effect of antioxidants on post-thawed Angora goat (Capra hircus ancryrensis) sperm parameters, lipid peroxidation and antioxidant activities. Small Rumin. Res. 89, 24-30.
ce p
Bucak, M.N., Sariözkan, S., Tuncer, P.B., Ulutaş, P.A., Akcadag, H.A., 2009a: Effects of antioxidants on microscopic semen parameters, lipid peroxidation and antioxidant activities in Angora goat semen following cryopreservation. Small Rumin. Res. 81, 90–95.
Ac
Bucak, M.N., Tekin, N., 2007. Protective effect of taurine, glutathione and trehalose on the liquid storage of ram semen. Small Rumin. Res. 73, 103-108. Bucak, M.N., Tuncer, P.B., Sariözkan, S., Ulutaş, P.A., 2009b: Comparison of the effects of glutamine and an amino acid solution on post-thawed ram sperm parameters, lipid peroxidation and anti-oxidant activities. Small Rumin. Res. 81, 13-17. Correa, J.R., Zavos, P.M., 1994. The hypoosmotic swelling test: its employment as an assay to evaluate
18
Page 18 of 33
the functional integrity of the frozen-thawed bovine sperm membrane. Theriogenology 42, 351-360. De Lamirande, E., Gagnon, C., 1999. The dark and bright sides of reactive oxygen species on sperm function. In: Gagnon, C. (Ed.), The Male Gamete: From Basic Science to Clinical Application. Cache
ip t
River Press, Vienna, Austria, pp. 455–467. De vries, J.E., Vork, M.M., Roemen, T.H.M., De jong, Y.F., Cleutjens, J.P.M. , Van der vusse, G.J., Van
neonatal rat ventricular myocytes. J. Lipid Res. 38, 1384–1394.
cr
bilsen, M., 1997. Saturated but not mono-unsaturated fatty acids induce apoptotic cell death in
us
Eslami, M., Ghaniei, A., Mirzie Rad, H., 2016. Effect of the rooster semen enrichment with oleic acid on the quality of semen during the chilled storage. Poult. Sci. 95, 1418-24.
an
Evans, G., Maxwell, W.M.C., 1987. Frozen storage of semen. In: Salamon’s Artificial Insemination of Sheep and Goats. Butterworths, Wellington 122–141.
M
Foulkes, J.A., 1977. The separation of lipoproteins from egg yolk and their effect on the motility and integrity of bovine sperm. J. Reprod. Fertil. 49, 277-284.
d
Frederick, S., 2010. Hep G2 Hepatocyte Lipid Peroxidation Assay. NCL Method GTA- 4.Version1.1.
te
Gandini, L., Lombardo, F., Paoli, D., Caponecchia, L., Familiari, G., Verlegia, C., 2000. Study of apoptotic DNA fragmentation in human spermatozoa. Hum. Reprod. 15, 830–839.
ce p
Graham, J.K., Foote, R.H., 1987. Effect of several lipids, fatty acyl chain length, and degree of unsaturation on the motility of bull sperm after cold shock and freezing. Cryobiology 24, 42-52. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982.
Ac
Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal. Biochem. 126, 131–138. Guerra, M.M.P., Evans, G., Maxwell, W.M.C., 2004. The role of oxidants and antioxidants on andrology. Rev. Bras. Reprod. Anim. 28, 187–195. Gundogan, M., Yeni, D., Avdatek, F., Fidan, A.F., 2010. Influence of sperm concentration on the motility, morphology, membrane and DNA integrity along with oxidative stress parameters of ram sperm during liquid storage. Anim. Reprod. Sci. 122, 200–207.
19
Page 19 of 33
King, M.E., McKelvey, W.A.C., Dingwall, W.S., Matthews, K.P., Gebbie, F.E., Mylne, M.J.A., Stewart, E., Robinson, J.J., 2004. Lambing rates and litter size following intrauterine or cervical insemination of frozen/thawed semen with or without oxytocin administration. Theriogenology 62, 1236–1244.
ip t
Koracevic, D., Koracevic, G., Djordjevic, V., Andrejevic, S., Cosic, V., 2001. Method for the measurement of antioxidant activity in human fluids. J. Clin. Pathol. 54, 356-361.
cr
Larsen, L., Scheike, T., Jensen, T.K., Bonde, J.P., Ernst, E., Hjollund, N.H., 2000. Computer-assisted semen analysis parameters as predictors for fertility of men from the general population. The Danish
us
First Pregnancy Planner Study Team. Hum. Reprod. 15, 1562–1567.
Ma, H., Quan, F., Chen, D., Zheng, Y., Zhang, B., Wang, Y., Zhang, Y., 2011. Protective function of
an
alpha-lipoic acid on sperm motility and mitochondrial function during goat sperm-mediated gene transfer. Small Rumin. Res. 99, 191-198.
M
Maedler, K., Oberholzer, J., Bucher, P., Spinas, G.A., Donath, M.Y., 2003. Monounsaturated fatty acids prevent the deleterious effects of palmitate and high glucose on human pancreatic beta-cell turnover
d
and function. Diabetes 52, 726–33.
te
Marklund, S., Marklund, G., 1974. Involvement of superoxide anion radical in the auto-oxidation of pyragallol, and a convenient assay for superoxide dismutase. Europ. J. Biochem. 47, 469 – 474.
ce p
Marti, J.I., Marti, E., Cebrian-Perez, J.A., Muino-Blanco, T., 2003. Survival rate and antioxidant enzyme activity of ram spermatozoa after dilution with different extenders or selection by a dextran swim-up procedure. Theriogenology 60, 1025-1037.
Ac
Maxwell, W.M.C., Stojanov, T., 1996. Liquid storage of ram semen in the absence or presence of some antioxidants. Reprod. Fertil. Dev. 8, 1013–1020. Maxwell, W.M.C., Watson, P.F., 1996. Recent progress in the preservation of ram semen. Anim. Reprod. Sci. 42, 55–65. Menendez, J.A., Papadimitropoulou, A., Vellon, L., Lupu, R., 2006. A genomic explanation connecting "Mediterranean diet", olive oil and cancer: Oleic acid, the main monounsaturated Fatty acid of olive
20
Page 20 of 33
oil, induces formation of inhibitory "PEA3 transcription factor-PEA3 DNA binding site" complexes at the Her-2/neu (erbB-2) oncogene promoter in breast, ovarian and stomach cancer cells. Eur. J. Cancer 42, 2425-2432.
ip t
Michael, A., Alexopoulos, C., Pontiki, E., Hadjipavlou-Litina, D., Saratsis, P., Boscos, C., 2007. Effect of antioxidant supplementation on semen quality and reactive oxygen species of frozen-thawed canine
cr
spermatozoa. Theriogenology 68, 204-212.
Moradi, A.R., Malekinejad, H., Farrokhi-Ardabili, F., Bernousi, I., 2013. Royal Jelly improves the sperm
us
parameters of ram semen during liquid storage and serves as an antioxidant source. Small Rumin. Res. 113, 346-352.
an
Narang, D., Sood, S., Thomas, M., Dinda, A., Maulik, S., 2004. Effect of dietary palm olein oil on oxidative stress associated with ischemic–reperfusion injury in isolated rat heart. BMC Pharmacol. 4,
M
29.
Oudit, G., Sun, H., Kerfant, B., Crackower, M., Penninger, J., Backx, P., 2004. The role of
d
phosphoinositide-3 kinase and PTEN in cardiovascular physiology and disease. J. Mol. Cell. Cardiol.
te
37, 449-471.
Parthasarathy, S., Khoo, J.C., Miller, E., Barnett, J., Witztum, J. L., Steinberg, D., 1990. Low density
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lipoprotein enriched in oleic acid is protected against oxidative modification: Implications for dietary prevention of atherosclerosis. Proc. Natl. Acad. Sci. USA 87, 3894-3898. Ruiz-Gutiérrez, V., Pérez-Espinosa, A., Vázquez, C.M., Santa-María. C., 1999. Effects of dietary fats
Ac
(fish, olive and high-oleic-acid sunflower oils) on lipid composition and antioxidant enzymes in rat liver. Br. J. Nut. 82, 233-241. Salamon, S., Maxwell, W.M.C., 2000. Storage of ram semen. Anim. Reprod. Sci. 62, 77-111. Sarıözkan, S., Bucak, M.N., Tuncer, P.B., Ulutas, P.A., Bilgen, A., 2009. The influence of cysteine and taurine on microscopic–oxidative stress parameters and fertilizing ability of bull semen following cryopreservation. Cryobiology 58, 134–138.
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Sheikholeslami Kandelousi, M.A., Arshami, J., Naserian, A.A., Abavisani, A., 2013. The Effect of addition omega-3, 6, 9, fatty acids on quality of bovine chilled and frozen-thawed sperms. Open Vet. J. 3, 47–52.
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Sicherle, C.C., Maia, M.S., Bicudo, S.D., Rodello, L., Azevedo, H.C., 2011. Lipid peroxidation and generation of hydrogen peroxide in frozen-thawed ram semen supplemented with catalase or Trolox.
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Small Rumin. Res. 95, 144–149.
Van Harken, D.R., Dixon, C.W., Heimberg, M., 1969. Hepatic Lipid Metabolism in Experimental
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Diabetes. J. Biol. Chem. 224, 2278-2285. 385
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386
Figure legends
388
Figure 1. Malondialdehyde (MDA) values (µmol/g protein; mean ± SE) in medium of rams following treatment with
389
different concentrations of oleic acid. A,B Values with different superscripts indicate difference (P < 0.05) among
390
groups at each time point. a,b,c,d Values with different superscripts indicate difference (P < 0.05) over time within the
391
experimental groups.
392
Figure 2. Malondialdehyde (MDA) values (µmol/g protein; mean ± SE) of ram spermatozoa following treatment
393
with different concentrations of oleic acid.
394
among groups at each time point.
395
within experimental groups.
396
Fig. 3. Total antioxidant capacity (TAC) (mmol/g protein; mean ± SE) in medium of rams following treatment with
397
different concentrations of oleic acid. A,B Values with different superscripts indicate significant difference (P < 0.05)
398
among groups at each time point.
399
within experimental groups.
400
Figure 4. Total antioxidant capacity (TAC) (mmol/g protein; mean ± SE) in spermatozoa of ram following treatment
401
with different concentrations of oleic acid.
Ac
ce p
te
d
M
387
a,b
a,b,c
A,B
Values with different superscripts indicate difference (P < 0.05)
Values with different superscripts indicate a difference (P < 0.05) over time
Values with different superscripts indicate a difference (P < 0.05) over time
A,B
Values with different superscripts indicate differences (P < 0.05)
22
Page 22 of 33
a,b,c,d
402
among groups at each time point.
403
within experimental groups.
404
Figure 5. Total amounts of nitric oxide (NO) (nmol/g protein; mean ± SE) in medium of rams following treatment
405
with different concentrations of oleic acid. There were no significant differences (P > 0.05) among groups at each
406
time point.
407
groups.
ip t
Values with different superscripts indicate differences (P < 0.05) over time within experimental
cr
a,b,c
Values with different superscripts indicate differences (P < 0.05) over time
us
408 409
Figure 6. Total amounts of nitric oxide (NO) (nmol/g protein; mean ± SE) in spermatozoa of ram following
410
treatment with different concentrations of oleic acid. A,B Values with different superscripts indicate differences (P <
411
0.05) among groups at each time point.
412
time within experimental groups.
413
Figure 7. Superoxide dismutase (SOD) activities (unit/mg protein; mean ± SE) in medium of rams following
414
treatment with different concentrations of oleic acid. There were no significant differences among groups at each
415
time point (P > 0.05).
416
experimental groups.
an
Values with different superscripts indicate differences (P < 0.05) over
M
a,b
d
Values with different superscripts indicate differences (P < 0.05) over time within
te
a,b
Figure 8. Superoxide dismutase (SOD) activities (unit/mg protein; mean ± SE) in spermatozoa of ram following
418
treatment with different concentrations of oleic acid. A,B Values with different superscripts indicate differences (P <
419
0.05) among groups at each time point.
420
time within experimental groups.
422
a,b
Values with different superscripts indicate differences (P < 0.05) over
Ac
421
ce p
417
423
23
Page 23 of 33
ip t
0.6
Control
Ad
cr
O 0.125
Bd Bd
0.4
Ac
Bd Bc
ABc ABc
Bc Bb
0.2
us
Ab Ab Ab Aa Ab Aa Aa Aa Aa Aa
0.0 Time 0h
O 0.25 O 0.5 O 1.0
an
Malondialdehyde (µmol/g protein)
Figure 1
Time 24h
Time 48h
Time 72h
te
d
M
Treatment Groups
ce p
1.0 0.8
Ac
Malondialdehyde (µmol/g protein)
Figure 2
Aa
0.6
Aa
Aa
Aa
Aa
Ab
ABaABa
Ab
Ab Ba Bab Ba
Ba Bab Ba
Control O 0.125
Ba Ba Bb Ba
O 0.25 O 0.5 O 1.0
0.4 0.2 0.0
Time 0h
Time 24h
Time 48h
Time 72h
Treatment Groups
Page 24 of 33
ip t
Figure 3
Ab
Aab Aa
O 0.125
Ab
BbcBbc Ba Bbc
Bc Ba Bc
Ab
1.5
Bc Ab
cr
2.0
Control
Aa Aa Aa Aa Aa
us
Ab
1.0 0.5 0.0 Time 0h
O 0.25 O 0.5 O 1.0
an
TAC (mmol/g protein)
2.5
Time 24h
Time 48h
Time 72h
te
d
M
Time of Experiment
4
2
Aa Aa Aa Aa Aa
ABb ABb Ab
Control O 0.125
Bb Bab Bbc Bbc ABc
Bb
O 0.25 Bd Bc Bb Bd
Abc
Ac
TAC (mmol/g protein)
6
ce p
Figure 4
O 0.5 O 1.0
Ac
0 Time 0h
Time 24h
Time 48h
Time 72h
Time of Experiment
Page 25 of 33
b
b c b
c b b
b
Control
b b
O 0.125
ab a b
O 0.25
ab
cr
ab
4
O 0.5
a a a
a
O 1.0
us
a
2
an
NO (nmol/g protein)
6
ip t
Figure 5
0 Time 0h
Time 24h
Time 48h
Time 72h
M
Treatment Groups
te
d
Figure 6
ce p
20
Ad
Aa
Aa
10
0
Aa
Aa
Aab Aa
Control O 0.125
Ac Bb Ba
Ab
Ba Ba Bb Bab
Ba Bb
O 0.25 O 0.5 O 1.0
Aa Aa Aa
Ac
NO (nmol/g protein)
30
Time 0h
Time 24h
Time 48h
Time 72h
Treatment Groups
Page 26 of 33
Control
30
O 0.125
a a a ab
20 a
a
a a b
b a
O 0.25
ab
O 0.5 O 1.0
a a a
cr
a
a ab
a
ip t
b
10
us
SOD activity (Unit/mg protein)
Figure 7
0 Time 0h
Time 24h
Time 48h
Time 72h
M
an
Treatment Groups
60
d te ce p
80
Aa Aa Aa Aa Aa
40 20 0
Ac
SOD activity (Unit/mg protein)
Figure 8
Time 0h
Control
Aab Aa Aa Aab Aa
Time 24h
Bb Bb Bb Ba
Bab Bab Bab Ba
O 0.125 O 0.25 O 0.5
Aab
O 1.0
Ab
Time 48h
Time 72h
Treatment Groups
Page 27 of 33
Table 4 Percentage of plasma membrane integrity test (HOST) of ram spermatozoa (mean ± SE) following enrichment with oleic acid and stored for various time points at 4 ◦C. Treatment
Time of storage (h) 24
48
Control
87.24 ± 1.79 Aa
76.68 ± 2.27 Ab
64.48 ± 2.00 Ac
O 0.125
87.55 ± 2.37 Aa
79.46 ± 2.32 Aa
68.79 ± 0.95 ABb
O 0.25
88.13 ± 1.84 Aa
82.50 ± 2.34 Aa
77.63 ± 2.39 BCab
70.43 ± 2.75 BCb
O 0.5
89.68 ± 2.25 Aa
83.39 ± 1.54 Aab
76.83 ± 1.81 BCbc
72.10 ± 2.63 BCc
O 1.0
88.77 ± 1.61 Aa
85.76 ± 1.63 Aab
78.73 ± 2.49 Cbc
us
cr
61.99 ± 1.51 ABb
74.53 ± 1.90 Cc
Values with different superscripts indicate differences (P < 0.05) among groups at each time point.
ce p
te
d
M
Values with different superscripts indicate significant differences (P < 0.05) between the data at the same row.
Ac
a,b,c
59.34 ± 2.12 Ac
an
A,B,C
72
ip t
0
Page 28 of 33
Table 3 Percentage of viable spermatozoa in ram semen (mean ± SE) following enrichment with oleic acid and stored for various time points at 4 ◦C. Treatment
Time of storage (h) 24
48
72
Control
95.32 ± 2.08 Aa
87.39 ± 3.71 Aab
79.77 ± 2.36 Abc
73.28 ± 1.60 Ac
O 0.125
95.26 ± 1.69 Aa
88.96 ± 2.81 Aab
81.03 ± 1.66 Abc
O 0.25
96.30 ± 1.30 Aa
91.87 ± 1.93 Ab
84.90 ± 2.75 Ac
O 0.5
93.23 ± 1.57 Aa
90.67 ± 1.67 Aa
85.28 ± 2.60 Ab
81.81 ± 2.29 Bb
O 1.0
94.12 ± 1.37 Aa
90.29 ± 1.70 Aab
85.78 ± 1.24 Ab
82.32 ± 2.36 Bb
78.76 ± 0.62 ABc
us
cr
80.76 ± 2.01 ABd
Values with different superscripts indicate difference (P < 0.05) among groups at each time point.
a,b,c,d
Values with different superscripts indicate significant differences (P < 0.05) between the data at the same
an
A,B
ip t
0
Ac
ce p
te
d
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Treatment
Time of storage (h) 0
24
48
72
VAP (µm/s)
Control O 0.125 O 0.25 O 0.5 O 1.0
39.12 ± 1.23 Aa 38.48 ± 1.47 Aa 39.73 ± 1.31 Aa 37.75 ± 2.54 Aa 37.42 ± 1.43 Aa
25.47 ± 1.63 Ab 33.19 ± 1.77 Aab 31.47 ± 0.78 Ab 31.95 ± 2.70 Aab 30.20 ± 2.17 Aab
20.70 ± 0.94 Abc 27.07 ± 1.97 Abc 25.24 ± 0.66 Ac 24.64 ± 1.34 Ab 23.81 ± 1.97 Ab
18.63 ± 1.15 Ac 23.88 ± 2.24 Ac 23.34 ± 1.81 Ac 24.66 ± 1.65 Ab 24.23 ± 1.70 Ab
VSL (µm/s)
Control
25.43 ± 4.23 Aa
15.50 ± 1.29 Aab
13.57 ± 0.38 Ab
12.14 ± 1.13 Ab
O 0.125 O 0.25
Aa
23.92 ± 5.03 24.19 ± 5.23 Aa
21.46 ± 1.78 23.00 ± 1.22 Aa
Aa
16.19 ± 1.84 16.54 ± 1.62 Aa
14.79 ± 1.37 Aa 15.27 ± 1.22 Aa
O 0.5 O 1.0
22.80 ± 4.27 Aa 24.15 ± 1.42 Aa
20.71 ± 1.35 Aa 19.02 ± 1.97 Aab
17.01 ± 1.91 Aa 17.09 ± 1.93 Ab
15.71 ± 0.61 Aa 16.54 ± 0.85 Ab
Control
118.95 ± 3.87 Aa
93.57 ± 2.67 Ab
63.58 ± 3.66 Ac
55.30 ± 2.13 Ac
O 0.125 O 0.25 O 0.5
Aa
117.97 ± 2.08 114.77 ± 3.90 Aa 119.08 ± 1.07 Aa
O 1.0
123.00 ± 3.25 Aa
Control O 0.125
75.01 ± 1.04 Aa 76.38 ± 5.30 Aa
O 0.25 O 0.5 O 1.0
73.05 ± 1.44 Aa 76.99 ± 3.19 Aa 74.57 ± 4.71 Aa
Control O 0.125 O 0.25 O 0.5 O 1.0
LIN (%)
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64.51 ± 1.65 Bc 69.71 ± 2.38 BCb 71.56 ± 2.30 BCc
108.63 ± 3.79 Ba
83.15 ± 3.50 Bb
77.85 ± 2.52 Cb
63.41 ± 1.86 Ab 66.00 ± 3.03 Aa
67.82 ± 1.09 Ab 65.04 ± 2.96 Aa
65.86 ± 2.54 Ab 65.48 ± 2.61 Aa
67.37 ± 1.84 Aa 73.28 ± 1.87 Aa 70.11 ± 2.63 Aa
68.83 ± 3.15 Aa 72.40 ± 1.89 Aa 69.29 ± 1.38 Aa
67.06 ± 2.17 Aa 70.34 ± 2.12 Aa 70.77 ± 2.10 Aa
29.16 ± 0.86 Aa 29.94 ± 2.27 Aa
20.58 ± 1.03 Ab 24.17 ± 2.41 Aa
21.23 ± 1.37 Ab 24.56 ± 0.78 Aa
20.44 ± 1.41 Ab 23.28 ± 1.78 Aa
28.96 ± 5.87 Aa 28.65 ± 1.21 Aa 30.11 ± 1.87 Aa
23.88 ± 1.80 Aa 25.88 ± 1.57 Aa 25.36 ± 2.57 Aa
23.78 ± 4.57 Aa 26.37 ± 1.22 Aa 23.22 ± 1.76 Aa
23.21 ± 1.44 Aa 25.32 ± 1.79 Aa 24.58 ± 1.51 Aa
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67.90 ± 2.76 75.57 ± 5.39 ABb 78.06 ± 1.95 Bc
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ABc
Values with different superscripts indicate differences (P < 0.05) among groups at each time point.
Ac
A,B,C
100.50 ± 0.81 102.33 ± 1.87 ABa 106.68 ± 3.16 Bb
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STR (%)
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VCL (µm/s)
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Parameter
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Table 2 Spermatozoa motion parameters of ram (mean ± SE) assessed by CASA following enrichment with oleic acid and stored for various time points at 4 ◦C.
Values with different superscripts indicate significant differences (P < 0.05) among various time points in a group.
VCL: curvilinear velocity (µm/s), VSL: straight-line velocity (µm/s), VAP: average path velocity (µm/s), LIN: linearity ([VSL/VCL]×100, %), STR: straightness ([VSL/VAP]×100, %).
Page 30 of 33
Table 1 Percentage of total and forward progressive motility of spermatozoa (mean ± SE) in ram semen following enrichment with oleic acid and stored for various time points at 4 ◦C. Parameter
Treatment
Time of storage (h) 24
48
72
Control
93.61 ± 0.79 Aa
85.49 ± 1.58 Ab
77.50 ± 1.31 Ac
70.49 ± 1.83 Ad
O 0.125
91.89 ± 1.41 Aa
89.46 ± 1.56 Aa
80.56 ± 0.77 ABb
77.01 ± 1.20 Bb
O 0.25
93.20 ± 1.87 Aa
89.80 ± 0.98 Aa
85.51 ± 1.16 Bab
78.78 ± 1.73 Bb
O 0.5
94.19 ± 1.50 Aa
90.97 ± 1.40 Aab
84.60 ± 0.77 Bbc
80.93 ± 1.51 Bc
O 1.0
91.42 ± 1.40 Aa
88.09 ± 1.65 Aab
84.11 ± 1.73 Bab
81.58 ± 1.28 Bb
Forward progressive
Control
89.37 ± 1.18 Aa
58.29 ± 1.25 Ab
49.71 ± 1.28 Ac
43.77 ± 1.54 Ac
motility
O 0.125
88.64 ± 1.41 Aa
62.31 ± 1.22 ABb
56.33 ± 1.62 ABbc
52.37 ± 1.42 Bc
O 0.25
88.71 ± 2.20 Aa
63.35 ± 1.15 ABb
58.56 ± 1.86 Bbc
53.31 ± 1.13 Bc
O 0.5
87.75 ± 1.58 Aa
67.43 ± 2.11 Bb
61.89 ± 1.73 Bbc
56.53 ± 1.67 Bc
O 1.0
88.03 ± 1.88 Aa
68.46 ± 1.64 Bb
63.12 ± 1.79 Bbc
57.26 ± 2.13 Bc
cr
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M
Values with different superscripts indicate difference (P < 0.05) among groups at each time point.
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Values with different superscripts indicate significant differences (P < 0.05) between the data at the same row.
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a,b,c,d
Ac
A,B
d
Total motility
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Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or
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organizations that could inappropriately influence or bias the content of the paper.
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Ram semen enrichment with oleic acid decreased the amounts of malondialdehyde and nitric oxide of spermatozoa during low temperature liquid storage. Moreover, it increased the total antioxidant capacity levels and superoxide dismutase activities of ram spermatozoa. Ultimately,
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addition of oleic acid to ram semen enhanced the kinematics of spermatozoa during storage at
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refrigerator temperature.
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S0921-4488(17)30060-3 http://dx.doi.org/doi:10.1016/j.smallrumres.2017.03.002 RUMIN 5440
To appear in:
Small Ruminant Research
Received date: Revised date: Accepted date:
22-9-2016 2-3-2017 3-3-2017
Please cite this article as: Hashem, E.Z., Haddad, R., Eslami, M.,Evaluation of ram semen enrichment with oleic acid on different spermatozoa parameters during low temperature liquid storage, Small Ruminant Research (2017), http://dx.doi.org/10.1016/j.smallrumres.2017.03.002 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.
Title: Evaluation of ram semen enrichment with oleic acid on different spermatozoa parameters
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during low temperature liquid storage
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Elham Zadeh Hashem1*, Roya Haddad2, Mohsen Eslami2
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Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
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Department of Theriogenology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
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Running Title: Ram semen enrichment with oleic acid
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*To whom correspondence should be addressed: Elham Zadeh Hashem, D.V.M., Ph.D.,
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Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
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Tel: +984432774737 ; Fax: +984432777099 ; E-mail: [email protected]
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ABSTRACT
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The purpose of the current experiment was to evaluate the protective effect of oleic acid on ram
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spermatozoa parameters during storage at refrigerator temperature. Ejaculates were collected
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from rams, pooled, diluted with extender and enriched with 0 (control), 0.125 (O 0.125), 0.25 (O
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0.25), 0.5 (O 0.5) and 1 (O1.0) mM oleic acid at a final concentration of 500×106
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spermatozoa/mL. Spermatozoa kinematics, viability and spermatozoa membrane integrity were
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evaluated by using computer-assisted sperm analysis (CASA), eosin-nigrosin staining and hypo-
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osmotic swelling (HOS) test, respectively. In addition, amounts of malondialdehyde (MDA),
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total antioxidant capacity (TAC), nitric oxide (NO) and superoxide dismutase (SOD) activities
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were assessed in the medium and spermatozoa at 0, 24, 48 and 72 h of storage. Enrichment of
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semen with oleic acid resulted in a significant (P < 0.05) increase in total motility, forward
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progressive motility and VCL (curvilinear velocity, µm/s) of O 0.5 and O1.0 groups at 48 and 72
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h of storage. In addition, viability and spermatozoa membrane functionality were greater in O 0.5
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(81.81 ± 2.29 % and 72.10 ± 2.63 %) and O1.0 (82.32 ± 2.36 % and 74.53 ± 1.90 %) groups
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compared to the control group (73.28 ± 1.60 % and 59.34 ± 2.12 %) at 72 h (P < 0.05). The
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measurement of MDA levels in spermatozoa showed that, there were higher in control group
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(0.84 ± 0.02 and 0.86 ± 0.04 µmol/g protein) in comparison with the O 0.125 (0.67 ± 0.05 and
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0.69 ± 0.03 µmol/g protein), O 0.25 (0.62 ± 0.03 and 0.66 ± 0.02 µmol/g protein), O 0.5 (0.62 ±
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0.02 and 0.65 ± 0.06 µmol/g protein) and O1.0 (0.60 ± 0.01 and 0.61 ± 0.01 µmol/g protein)
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groups at 48 and 72 h of storage (P = 0.002). Moreover, higher amounts of TAC were obtained
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in oleic acid treated groups compared to control group in spermatozoa and medium at 48 and 72
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h (P < 0.05). Nitrosative status was lower and SOD activities were higher in spermatozoa of oleic
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acid treated groups than the control group at 48 and 72 h (P < 0.05). Our results indicated that
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oleic acid with the dose of 0.5 and 1 mM would enhances ram semen quality during liquid
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storage at 48 and 72 h.
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Keywords: Oleic acid, Ram semen, Sheep, Total antioxidant capacity, Malondialdehyde.
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1. Introduction
One of the most important domestic species due to having triple productive profiles of milk,
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lamb and wool are sheep (Evans and Maxwell, 1987). Artificial insemination (AI), which is
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performed by using fresh or cooled diluted semen in sheep, makes the dissemination of genetic
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material from a small number of superior males to a large number of females possible (Maxwell
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and Watson, 1996). Storage of diluted semen has been widely used in AI programs. When the
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insemination is done within a short period of time after collection, diluted and cooled ram semen
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is a practical alternative to frozen semen to avoid freezing/thawing induced injuries (King et al.,
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2004). In small ruminants, the success of the AI technique depends on various factors especially
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those related with the semen processing and preservation.
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The temperature at which lipids change from the fluid to the crystalline state named phase
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transition temperature of plasma membrane (Foulks, 1977). Long time storage of spermatozoa
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above freezing point causes membrane deterioration, because of membrane phase transition
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occurring in the spermatozoa plasma membrane (Graham and Foot, 1987). One of the causes of
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decreased sperm viability following semen storage has been stated as the production of reactive
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oxygen species (ROS), which are generated from the metabolism of oxygen (O2) (Aitken et al.,
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1993). The mammalian spermatozoa is a redox active cell, which generates ROS spontaneously.
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Excessive ROS which is produced during liquid semen storage initiates oxidative reactions that
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eventually lead to the death of the sperm cells (De Lamirande and Gagnon, 1999; Guerra et al.,
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2004). To protect the spermatozoa against the harmful effects of the lipid peroxidation (LPO),
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several researches have been made to enrich semen by adding substances such as taurine, royal
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jelly, SOD, catalase, trehalose, glutathione, glutathione peroxidase, cytochrome c, glutamine and
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hyaluronan to ram semen (Maxwell and Stojanov, 1996; Bucak and Tekin, 2007; Bucak et al.,
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2009b; Moradi et al., 2013). Nevertheless, improvement in semen quality during cold storage
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still appears to be a challenge in sheep industry.
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Beneficial effects of oleic acid, the n-9 monounsaturated fatty acid with a 18-carbon chain, on
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total anti-oxidant capacity (TAC) and signal transduction alteration during in vitro (De Vries et
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al., 1997; Maedler et al., 2003; Menendez et al., 2006; Eslami et al., 2016; on rat ardiomyocytes,
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human pancreatic beta-cell, human breast, ovarian and stomach cancer cells and rooster sperm,
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respectively) and in vivo (Parthasarathy et al., 1990; Ruiz-Gutiérrez et al., 1999, on rabbit and
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rat, respectively) experiments have been well documented. However, the effect of oleic acid on
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ram semen during liquid storage has not yet been reported. Therefore, the current trial was
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performed to evaluate the effect of oleic acid on spermatozoa motion characteristics (evaluated
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by computer assisted sperm analysis; CASA), viability, spermatozoa plasma membrane
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functionality, amounts of malondialdehyde (MDA), TAC, nitric oxide (NO) and superoxide
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dismutase (SOD) activity of ram semen during low temperature liquid storage.
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2. Materials and Methods
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2.1. Animals and semen collection
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Animals were housed at the Research Farm of Agriculture Faculty of Urmia University,
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Urmia, Iran (Nazloo campus, East longitude 44◦, 58׳, 30 ״and North latitude 37◦, 39׳, 30 )״and
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maintained under uniform nutritional conditions (free access to good quality of hay and water).
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Ejaculates were collected from five mature Qezel rams (3 to 4 years of age; mean body weight=
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62.5 ± 3.1), twice a week with the aid of an artificial vagina in the presence of estrus ewes during
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the winter (four weeks; January 2016 to February 2016). Immediately after collection, semen
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was transferred to the laboratory and placed in a water bath (37 ˚C) for semen evaluation. The
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semen samples were evaluated for volume, sperm concentration, and percentage of motility.
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Only ejaculates between 0.75-2 mL volume with a concentration of greater than 2.5 × 109
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sperm/mL having >80% forward progressive motile sperm were selected and pooled for further
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analysis. Seven pooled ejaculates (35 individual semen samples; each ram gave seven samples)
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were used in the present experiment. The Animal Care committee of the Urmia University
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approved the semen sampling procedure.
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2.2. Semen processing and evaluation
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After pooling, semen was diluted using a tris-based extender containing of Tris
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(hydroxymethyl aminomethane) 3.63 g, fructose 0.5 g, citric acid 1.99 g, egg yolk 14% (v/v),
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100,000 IU penicillin and 100 mg streptomycin dissolved in distilled water (up to 100 mL), to a
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final concentration of 500×106 sperm/mL was used for medium of spermatozoa (Salamon and
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Maxwell, 2000). Each pooled semen sample was divided into 5 equal aliquots to treat with
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different concentrations of the oleic acid (1 ml for each treatment group at each time point). Both
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extender (medium) and semen were at 37◦C when mixed. Then, diluted semen was enriched with
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0 (control), 0.125 (O 0.125), 0.25 (O 0.25), 0.5 (O 0.5), and 1 (O1.0) mmolar bovine serum
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albumin (BSA)-conjugated oleic acid. Following enrichment, the semen was stored for 72 h at
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4◦C. The CASA (Test Sperm 2.1; Videotest, St. Petersburg, Russia) parameters (including total
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and forward progressive motility: FPM), percentage of viability and percentage of spermatozoa
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with healthy membrane were evaluated at 0, 24, 48 and 72 h of storage. Moreover, amounts of
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TAC, MDA, NO, activity of SOD and protein were measured in the medium and the
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spermatozoa at the mentioned time points. Oleic acid was purchased from Sigma Company (O
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1008, Sigma-Aldrich) and other materials used in this project were bought from the Merck
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Company.
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2.3. Preparation of BSA conjugated oleic acid
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Oleic acid was conjugated with BSA according to the method described by Van Harken et al.
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(1969) for the better utilization of oleic acid by spermatozoa. In brief, BSA solution (24%) was
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prepared in NaCl 0.09%. Then, 0.12g oleic acid was dissolved in 3 ml ethanol 95% and
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neutralized with NaOH. Then dissolved 0.13 g Na-oleate in 15 ml sodium chloride (0.9%). The
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Na-oleate was subsequently mixed with BSA solution and stored in the freezer -20 °C until used
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in the study.
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2.4. Spermatozoa kinematics evaluated by CASA
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A computer assisted sperm analyzer was used to assess spermatozoa kinematics. This system
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included a phase contrast microscope (Olympus, BX41, Tokyo, Japan) with a stage warmer, a
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CCD-camera (SDC-313B, Samsung Techwin Co., Gyeong, Korea). Videotaping of sperm
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motility was conducted at a rate of 25 frames/s. To assess spermatozoa kinematics, aliquot of
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extended semen was more diluted (25×106 cell/mL) with the same extender in each group. A 10
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µL of extended semen was placed on a warmed (37 ◦C) slide and covered with a cover slip
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(22mm×22 mm). The motion characteristics of spermatozoa was assessed at 37 ◦C at 200×.
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For each sample seven to eight fields per drop were analyzed and a minimum of 200
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spermatozoa were evaluated. The sperm variables included in the analysis, were total motility,
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FPM, curvilinear velocity (VCL, µm/s), straight-line velocity (VSL, µm/s), average path velocity
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(VAP,
µm/s),
linearity
(LIN
=
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[VSL/VAP]×100, %).
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2.5. Spermatozoa viability assessment
[VSL/VCL]×100,
%)
and
straightness
(STR
=
The percentage of viability of spermatozoa in the control and oleic acid treated groups was
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assessed by means of eosin-nigrosin staining (Evans and Maxwell, 1987). The final composition
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of the stain was: eosin-Y 1.67 g, nigrosin 10 g, and sodium citrate 2.9 g, dissolved in distilled
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water (up to 100 mL). Spermatozoa suspension smears were prepared by mixing a 5µL of the
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semen sample with 10 µL of the stain on a warm slide and spreading the stain with a second
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slide. The viability was assessed by counting at least 200 cells under light microscope (1000×,
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oil immersion). Spermatozoa showing partial or complete purple color was considered non-
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viable and spermatozoa showing no color, indicating the complete exclusion of the stain were
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considered to be alive.
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2.6. Assessment of spermatozoa membrane functionality by using the hypo-osmotic swelling
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(HOS) test
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Spermatozoa membrane integrity was assessed by HOS test consisting of fructose (0.9% w/v)
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and sodium citrate solution (0.49% w/v, 100 mOsm/kg), which was prepared on a daily basis and
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kept at 4°C (Correa and Zavos, 1994). Diluted semen (20 µl) was added to the pre-warmed
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(37°C) HOS solution (200 µl) in a test tube and then incubated for 60 min at 37°C. Hypo-
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osmotic swelling test was undertaken under a phase contrast microscope (400×, Olympus, BX41,
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Tokyo, Japan), counting 200 sperms, to determine the percentage of spermatozoa with
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curled/swollen tail.
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2.7. Separation of spermatozoa from medium
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The spermatozoa and the medium were separated by centrifugation. Semen was centrifuged
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for 10 minutes at 550 g. The resulting pellet was used as the concentrated spermatozoa aliquot
158
for purposes of the present study. The supernatant was centrifuged two more times, first for 10
159
minutes at 550 g, and then for 30 minutes at 3000 g. The resulting supernatant was considered to
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be the medium for purposes of the present study (Blesbois et al., 1993). The pellet of
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spermatozoa was re-suspended in 1 ml phosphate buffer saline (PBS), then trichloroacetic acid
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50% was added to the PBS to break the spermatozoa membrane.
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2.8. Amounts of MDA in the spermatozoa and medium
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Amounts of MDA were measured using the thiobarbituric acid reaction, using the method
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described by Frederick (2010). Briefly, the spermatozoa suspension or medium was added to
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thiobarbituric solution (one volume sample to two volume solution), mixed, and then double
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distilled water was added and the mixture was shaken. Tubes were heated in boiling water, then
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cooled and centrifuged for 10 minutes at 1000 g. The absorbance of the upper layer was read at
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532 nm wave length. The amounts of MDA were expressed as µmol/g protein in the
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spermatozoa and medium samples.
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2.9. Amounts of TAC in spermatozoa and medium
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Amounts of TAC were measured using the methods of Koracevic et al. (2001) with a slight
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modification. In brief, 490 µl of PBS solution were added to 10 µl of the sample. Additionally,
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sodium benzoate, acetic acid, Fe-EDTA and H2O2 were added to the tubes, respectively. The
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tubes were incubated at 37 °C for 60 minutes. Acetic acid and thiobarbituric solution were
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subsequently added to the tubes. The tubes were incubated in 100 °C water bath for 10 minutes.
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The absorbance of the samples was measured at 532 nm wave length. Amounts of TAC were
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expressed as mmol/g protein in the spermatozoa and medium samples.
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2.10. Amounts of total NO in spermatozoa and medium The total NO values in the spermatozoa and medium were measured according to the Griess
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reaction (Green et al., 1982). In Griess reaction nitric oxide rapidly converted into the more
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stable nitrite, which in an acidic environment nitrite is converted to HNO2. In reaction with
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sulphanilamide, HNO2
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ethylenediamine·2HCl to form an azo dye that can be detected by absorbance at a wavelength of
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540 nm. The NO values were expressed as nmol/g protein in the spermatozoa and medium
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samples.
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2.11. Measuring the SOD activities in spermatozoa and medium
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forms a diazonium salt, which reacts with N-(1-naphthyl)
The SOD activities was evaluated by pyrogallol oxidation method. The total SOD activity
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was determined according to Marklund and Marklund (1974), assaying the auto oxidation and
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illumination of pyrogallol at 420 nm for 3.5 min. One unit total SOD activity was calculated as
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the amount of protein causing 50% inhibition of pyrogallol autooxidation. The total SOD activity
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was expressed as units per milligram of protein in the spermatozoa and medium samples.
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2.12. Total protein measurement in the spermatozoa and medium
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Total protein was measured according to Bradford protocol (1976). In brief, Bradford reagent
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and stock solution of bovine serum albumin were prepared. The Bradford reagent was
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subsequently added to the spermatozoa and medium samples. The absorbance of the contents of
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the test tubes was measured at 595 nm wave length after 15 minutes. Amounts of total protein
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were obtained using a standard curve developed for this purpose.
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2.13. Statistical analysis
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Values of spermatozoa kinematics, viability, spermatozoa membrane functionality and
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amounts of TAC, MDA, NO and SOD activities were expressed as the mean ± SEM.
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Percentage data were subjected to arcsine transformation. Means were analyzed using a one-way
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analysis of variance, followed by a Tukey’s post hoc test to determine significant differences in
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all the variables recorded among groups. Changes of the variables over time were analyzed using
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a Repeated Measure ANOVA to reveal the differences among different time points in each
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treatment group. All analyses were done using SigmaStat software (Version 3.5; Chicago, IL).
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For all statistical analyses, differences with P < 0.05 were considered significant.
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3. Results
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3.1. Spermatozoa motility variables assessed by CASA
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There were no significant differences in total motility among groups at 0 h and 24 h (P > 0.05;
211
Table 1), while it was greater in O 0.25, O 0.5 and O1.0 groups compared to the control group at
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48 h of storage (Table 1; P = 0.004). Moreover, total motility was greater in oleic acid treated
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groups compared to control group at 72 h of storage (Table 1; P = 0.003). Within group analysis
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indicated that total motility were less at 72 h compared to 0 h in all treated groups (P < 0.023;
215
Table 1). There were no significant differences in FPM among groups at 0 h (Table 1; P > 0.05).
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But, it was greater in O 0.5 and O1.0 groups compared to control groups at 24 h, 48 h and 72 h of
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storage (Table 1; P < 0.005). Moreover, FPM was greater in O 0.25 compared to control at 48 h,
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and O 0.125 and O 0.25 compared to control at 72 h of storage (Table 1; P < 0.002). Within
219
group analyses revealed that the FPM was less at 24 h compared to 0 h, and 72 h compared to 24
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h in all groups (Table 1; P < 0.001). The VCL variable was significantly higher in oleic acid
221
treated groups compared to control group at 72 h of storage (P < 0.001; Table 2). There were no
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significant differences between oleic acid treated groups and control group at other variables
223
assessed by CASA (P > 0.05; Table 2).
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3.2. Viability of spermatozoa
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The interaction between time of storage and treatment indicated that the percent of viability
226
did not differ among treatment groups at 0 h, 24 h and 48 h (P > 0.05; Table 3), but the percent
227
of viability was greater in O 0.5 and O1.0 groups compared to control at 72 h of storage (P=
228
0.04; Table 3). Within group analysis revealed that the percent of viability was less at 48 h and
229
72 h compared to 0 h in all treated groups (P < 0.013; Table 3).
230
3.3. Hypo-osmotic swelling test of spermatozoa
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The result of the present experiment revealed that oleic acid at proper concentration was able
232
to protect plasma membrane of spermatozoa from damages during long time liquid storage (72h).
233
There were no significant differences among groups in percent of spermatozoa with intact
234
plasma membrane at 0 h and 24 h of storage (Table 4; P > 0.05). While, the percent of
235
spermatozoa with intact plasma membrane was greater in O 0.25, O 0.5 and O1.0 groups
236
compared to control group at 48 h and 72 h of storage (Table 4; P < 0.003). Changes over time
237
indicated that the percent of spermatozoa with intact plasma membrane was lower at 72 h
238
compared to 0 h in all treated groups (Table 4; P < 0.009).
239
3.4. Malondialdehyde values (µmol/ g protein)
240
3.4.1. Medium
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The interaction between time of storage and treatment revealed that the amounts of MDA did
242
not differ among the treatment groups at 0 h and 24 h of storage (Fig. 1, P > 0.05), while MDA
243
values were less in O 0.5 and O1.0 groups compared to control group at 48 h (Fig. 1, P = 0.041).
244
Moreover, MDA values were greater in control group compared to O 0.125, O 0.25, O 0.5 and
245
O1.0 groups at 72 h of storage (Fig. 1, P < 0.001). Within group analysis showed that the MDA
246
values were increased during the experiment, and these increases were significant in all treated
247
groups between 0 h and 48 h, and 72 h and 48 h of storage (Fig. 1, P < 0.001).
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248
3.4.2. Spermatozoa The results indicated that the MDA values (µmol/ g protein) of spermatozoa did not differ
250
among the treatment groups at 0 h (Fig. 2, P > 0.05). Whereas, the amounts of MDA were less in
251
O 0.5 and O1.0 groups compared to the control group at 24 h (Fig. 2, P = 0.016). Moreover, the
252
values of MDA were significantly less in oleic acid treated groups compared to control group at
253
48 h and 72 h of storage (Fig. 2, P = 0.002). Within group analysis revealed that amounts of
254
MDA in spermatozoa were greater at 24 h, 48 h and 72 h compared to 0 h in control group (Fig.
255
2, P = 0.002). Moreover, the MDA values were greater at 72 h compared to 0 h in O 0.5 group
256
(Fig. 2, P = 0.046). There were no significant differences in O 0.125, O 0.25 and O1.0 groups
257
among different time points (Fig. 2, P > 0.05).
258
3.5. Total TAC (mmol/ g protein)
259
3.5.1. Medium
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Amounts of TAC were not differ among groups at 0 h and 24 h (Fig. 3, P > 0.05). While,
261
amounts of TAC were greater in oleic acid treated groups compared to the control group at 48 h
262
and 72 h of storage (Fig. 3, P = 0.003). Within group analysis revealed that TAC values were
263
lower at 48 h and 72 h compared to 0 h in control, O 0.125, O 0.25 and O1.0 groups (Fig. 3, P <
264
0.004), whereas, there were no significant differences among different time points in O 0.5 group
265
(P = 0.112; Fig. 3).
266
3.5.2. Spermatozoa
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The interaction between time of storage and treatment indicated that significant differences
268
were not observed for amounts of TAC among groups at 0 h (Fig. 4; P > 0.05). But, values of
269
TAC were higher in O 0.5 (4.27 ± 0.16) and O1.0 (4.34 ± 0.13) groups compared to control (3.59
270
± 0.18) group at 24 h of storage (Fig. 4; P = 0.036). Moreover, amounts of TAC were greater in
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O 0.25 (3.72 ± 0.10), O 0.5 (3.64 ± 0.15) and O1.0 (3.75 ± 0.25) groups compared to control
272
(2.91 ± 0.16) at 48 h (Fig. 4; P = 0.030). On the other hand, the values of TAC were higher in
273
oleic acid treated groups compared to control group at 72 h of storage (Fig. 4; P < 0.001). Within
274
group analysis showed that the amounts of TAC in spermatozoa were less at 48 h and 72 h
275
compared to 0 h in all treated groups (Fig. 4, P < 0.008).
276
3.6. Total NO (nmol/g protein)
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3.6.1. Medium
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There were no significant differences in total amounts of NO among treatment groups at each
279
time point (P > 0.05; Figure 5). Within group analysis showed that the levels of NO were greater
280
at 48 h and 72 h compared to 0 h in all treated groups (P < 0.014; Fig. 5).
281
3.6.2. Spermatozoa
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The interaction between time of storage and treatment showed that there were no significant
283
differences for amounts of NO among groups at 0 h and 24 h, while amounts of NO were
284
significantly less in oleic acid treated groups compared to the control group at 48 h and 72 h (P <
285
0.011; Figure 6). Within group analysis indicated that the amounts of NO were significantly
286
increased in a time dependency manner in the control group (Fig. 6; P < 0.001). Moreover,
287
amounts of NO were greater at 72 h than the 0 h in O 0.5 and O1.0 groups (Fig. 6; P < 0.022).
288
But, there were no significant differences among different time points in O 0.125 and O 0.25
289
groups (Fig. 6; P > 0.05).
290
3.7. Superoxide dismutase (unit/mg protein)
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3.7.1. Medium
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The measuring of SOD activities indicated that there were no significant differences among
293
groups at each time point (P > 0.05; Figure 7). Within group analysis revealed that SOD
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activities were greater at 48 h than the 0 h in O 0.25 and O1.0 groups (Fig. 7; P < 0.037), but
295
significant differences were not observed among different time points in other groups (Fig. 7; P >
296
0.05).
297
3.7.2. Spermatozoa
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The interaction between time of storage and treatment showed that there were no significant
299
differences for SOD activities among groups at 0 h and 24 h (Fig. 8; P > 0.05), while SOD
300
activities were greater for oleic acid treated groups than the control group at 48 h and 72 h of
301
storage (Fig. 8; P < 0.011). Changes over time indicated that the activities of SOD were higher at
302
72 h compared to 0 h in O 0.125, O 0.25 and O 0.5 (Fig. 8; P < 0.039), while there were lower at
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72 h compared to 0 h in control group (Fig. 8; P = 0.005).
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4. Discussion
Our experiment showed that oleic acid at the proper concentration and varying with time of
307
storage was able to promote total motility, forward progressive motility, VCL, percent of
308
viability, percent of spermatozoa with healthy plasma membrane, and amounts of total
309
antioxidant capacity and superoxide dismutase activities compared to control group. Moreover,
310
oleic acid ameliorates amounts of malondialdehyde and nitric oxide of ram spermatozoa during
311
low temperature liquid storage. To our knowledge, it the first report of oleic acid on different
312
spermatozoa parameters in small ruminants.
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In the present study, supplementation of ram semen with 0.5 and 1 mM oleic acid resulted in
314
14% and 16% increase in VCl at 24 h, 23% and 31% increase at 48 h and 29% and 40% increase
315
at 72 h compared to the control semen, respectively. On the other hand, enrichment of ram
316
semen with 0.5 and 1 mM oleic acid caused 16 % and 17% increase in FPM compared to the
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control semen at 24 h, 24% and 27% increase at 48 h and 29% and 30% increase at 72 h,
318
respectively. Then, oleic acid was able to improve the ram spermatozoa VCL at proper
319
concentration in according to time of storage. Moreover, adding of oleic acid to the ram semen at
320
the 0.5 and 1 mM caused more than 10% increase in viability at 72 h of storage. It has been
321
demonstrated that the fertilizing capacity of spermatozoa was correlated with quantitative
322
assessment of spermatozoa motility by CASA (Larsen et al., 2000). Moreover, among the
323
spermatozoa kinematics, VCL has been shown as the most significant and independent CASA
324
parameter, which highly correlates with the fertilization rate (Larsen et al., 2000). It seems that,
325
monounsaturated fatty acids could strengthen the antioxidant defense and PI3 kinase levels,
326
which are of the important factors in the viability of the cells (Oudit et al., 2004). Previous
327
studies indicated that semen supplementation with antioxidants resulted in higher viability and
328
motility of small ruminant spermatozoa [ram (Maxwell and Stojanov, 1996; Bucak et al., 2008),
329
goat (Bucak et al., 2010)]. However, bull semen enrichment with n-3 fatty acids and soft gels
330
containing n-3, n-6, n-9 fatty acids reduced the spermatozoa motility and viability of chilled and
331
frozen-thawed semen (Abavisani et al. 2013; Sheikholeslami Kandelousi et al. 2013). The
332
present study also indicated that spermatozoa viability and motility decreased during the
333
experiment. The probable reason for the reduction of spermatozoa viability and motility during
334
the cold storage period could be related to the endogenously produced ROS. Previous study
335
showed that the cooling process, which we provided in the experiments by storing the samples at
336
4 ◦C, stimulated the ROS production in spermatozoa (Sicherle et al., 2011).
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337
The amounts of polyunsaturated fatty acids in the spermatozoa membranes in small ruminant
338
is generally higher than in other species. Then, the spermatozoa of small ruminant is highly
339
susceptible to oxidative damage during storage, resulting from the production ROS (Gandini et
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al., 2000). Because of lower antioxidant capacity, spermatozoa could not counteract the
341
damaging effects of ROS and LPO during liquid storage. Then, the application of antioxidant to
342
the semen is mandatory to reduce the harmful effects of LPO during storage. It has been shown
343
that enrichment of goat semen with taurine reduced the MDA levels compared to the control
344
(Ateşşahin et al., 2008). On the other hand, adding of alpha lipoic acid to the buck (Ma et al.,
345
2011) and dog (Michael et al., 2007) semen resulted in lower LPO. While, supplementation of
346
ram semen with glutathione, oxidized glutathione or cysteine did not affect the MDA levels
347
(Bucak et al., 2008). The present study indicated that, oleic acid was able to reduce the MDA
348
levels in medium and ram spermatozoa during storage at refrigerator. It seems that the efficacy
349
of antioxidant on the amounts MDA would depends on concentration of antioxidant, type of
350
antioxidant, time of storage and animal species.
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340
The current experiment revealed that ram semen enrichment with oleic acid increased the
352
amounts of TAC in spermatozoa and medium. The results of the present work are compatible
353
with the previous reports about the improvement of TAC following supplementation of ram
354
semen with royal jelly, superoxide dismutase, taurine and cysteine (Marti et al., 2003; Bucak and
355
Tekin, 2007; Bucak et al., 2008; Moradi et al., 2013). Whereas, our result were inconsistent with
356
the findings of Bucak et al. (2009a) where it was reported that administration of antioxidants
357
glutamine and hyaluronan to the goat semen did not affect the TAC levels. Previous studies
358
showed that the increase in antioxidant indices following the treatment with monounsaturated
359
fatty acids could be a result of their potency to elevate essential enzymatic antioxidants (Narang
360
et al., 2004). It has been reported that SOD activity was found to be higher in the presence of
361
curcumin and carnitine compared to the semen of the control group in goat (Bucak et al., 2010).
362
Also, elevated SOD activity was observed following supplementation of bull semen with
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cysteine (Sarıözkan et al. 2009). While, other antioxidants (Glutamin and hyaloronan) treatment
364
decreased the SOD activity in goat and ram semen (Bucak et al., 2009 a,b). Our result verify the
365
findings of previous work about the ability of monounsaturated fatty acids in the elevation of
366
SOD activities in treated cell (Narang et al., 2004). The results of the present work indicated that,
367
with the prolongation of storage time, amounts of TAC were decreased and MDA and NO levels
368
were increased. It is suggesting the key role of the oxidative/nitrosative stress in long time liquid
369
storage period. The results of the present experiment are compatible with the previous reports
370
about the detrimental effects of long liquid storage on the levels of generated MDA and NO
371
(Gundogan et al., 2010; Moradi et al., 2013).
372
5. Conclusion
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In conclusion, our results showed the protective effects of the oleic acid supplementation on
374
ram sperm viability and plasma membrane integrity during liquid storage condition. The current
375
study also showed that oleic acid decreased the amounts of malondialdehyde and nitric oxide,
376
increased the total antioxidant capacity levels and superoxide dismutase activities and ultimately
377
enhanced the kinematics of ram spermatozoa compared to the control during low temperature
378
liquid storage. It seems that, the protective effects of oleic acid at 48h and 72h likely related to its
379
antioxidant property, which could be provided at suitable concentrations (0.5 and 1 mM).
380
Acknowledgments
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The present experiment was funded by the Research Deputy of the Urmia University. We are
382
grateful to Dr. Farhad Farrokhi-Ardabili for his kind assistance during the semen sampling
383
process.
384
References Abavisani, A., Arshami, J., Naserian, A.A., Sheikholeslami Kandelousi, M.A., Azizzadeh, M., 2013.
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Quality of bovine chilled or frozen-thawed semen after addition of omega-3 fatty acids supplementation to extender. Int. J. Fertil. Steril. 7, 161–168. Aitken, R.J., Harkiss, D., Buckingham, D., 1993. Relationship between iron catalyzed lipid peroxidation
ip t
potential and human sperm function. J. Reprod. Fertil. 98, 257–265. Ateşşahin, A., Bucak, M.N., Tuncer, P.B., Kizil, M. 2008. Effects of anti-oxidant additives on
cr
microscopic and oxidative parameters of Angora goat semen following the freeze-thawing process. Small Rumin. Res. 77, 38-44.
us
Blesbois, E., Grasseau, I., Blum, J.C., 1993. Effects of Vitamin E on fowl semen storage at 4◦C. Theriogenology 39, 771–779.
an
Bradford, M.M., 1976. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 7, 248-254.
M
Bucak, M.N. Ateşşahin, A., Yüce, A., 2008. Effect of anti-oxidants and oxidative stress parameters on ram semen after the freeze-thawing process. Small Rumin. Res. 75, 128-134.
d
Bucak, M.N., Sariözkan, S., Tuncer, P.B., Sakin, F., Ateşşahin, A., Kulaksiz, R., Ҫevik, M., 2010. The
te
effect of antioxidants on post-thawed Angora goat (Capra hircus ancryrensis) sperm parameters, lipid peroxidation and antioxidant activities. Small Rumin. Res. 89, 24-30.
ce p
Bucak, M.N., Sariözkan, S., Tuncer, P.B., Ulutaş, P.A., Akcadag, H.A., 2009a: Effects of antioxidants on microscopic semen parameters, lipid peroxidation and antioxidant activities in Angora goat semen following cryopreservation. Small Rumin. Res. 81, 90–95.
Ac
Bucak, M.N., Tekin, N., 2007. Protective effect of taurine, glutathione and trehalose on the liquid storage of ram semen. Small Rumin. Res. 73, 103-108. Bucak, M.N., Tuncer, P.B., Sariözkan, S., Ulutaş, P.A., 2009b: Comparison of the effects of glutamine and an amino acid solution on post-thawed ram sperm parameters, lipid peroxidation and anti-oxidant activities. Small Rumin. Res. 81, 13-17. Correa, J.R., Zavos, P.M., 1994. The hypoosmotic swelling test: its employment as an assay to evaluate
18
Page 18 of 33
the functional integrity of the frozen-thawed bovine sperm membrane. Theriogenology 42, 351-360. De Lamirande, E., Gagnon, C., 1999. The dark and bright sides of reactive oxygen species on sperm function. In: Gagnon, C. (Ed.), The Male Gamete: From Basic Science to Clinical Application. Cache
ip t
River Press, Vienna, Austria, pp. 455–467. De vries, J.E., Vork, M.M., Roemen, T.H.M., De jong, Y.F., Cleutjens, J.P.M. , Van der vusse, G.J., Van
neonatal rat ventricular myocytes. J. Lipid Res. 38, 1384–1394.
cr
bilsen, M., 1997. Saturated but not mono-unsaturated fatty acids induce apoptotic cell death in
us
Eslami, M., Ghaniei, A., Mirzie Rad, H., 2016. Effect of the rooster semen enrichment with oleic acid on the quality of semen during the chilled storage. Poult. Sci. 95, 1418-24.
an
Evans, G., Maxwell, W.M.C., 1987. Frozen storage of semen. In: Salamon’s Artificial Insemination of Sheep and Goats. Butterworths, Wellington 122–141.
M
Foulkes, J.A., 1977. The separation of lipoproteins from egg yolk and their effect on the motility and integrity of bovine sperm. J. Reprod. Fertil. 49, 277-284.
d
Frederick, S., 2010. Hep G2 Hepatocyte Lipid Peroxidation Assay. NCL Method GTA- 4.Version1.1.
te
Gandini, L., Lombardo, F., Paoli, D., Caponecchia, L., Familiari, G., Verlegia, C., 2000. Study of apoptotic DNA fragmentation in human spermatozoa. Hum. Reprod. 15, 830–839.
ce p
Graham, J.K., Foote, R.H., 1987. Effect of several lipids, fatty acyl chain length, and degree of unsaturation on the motility of bull sperm after cold shock and freezing. Cryobiology 24, 42-52. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982.
Ac
Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal. Biochem. 126, 131–138. Guerra, M.M.P., Evans, G., Maxwell, W.M.C., 2004. The role of oxidants and antioxidants on andrology. Rev. Bras. Reprod. Anim. 28, 187–195. Gundogan, M., Yeni, D., Avdatek, F., Fidan, A.F., 2010. Influence of sperm concentration on the motility, morphology, membrane and DNA integrity along with oxidative stress parameters of ram sperm during liquid storage. Anim. Reprod. Sci. 122, 200–207.
19
Page 19 of 33
King, M.E., McKelvey, W.A.C., Dingwall, W.S., Matthews, K.P., Gebbie, F.E., Mylne, M.J.A., Stewart, E., Robinson, J.J., 2004. Lambing rates and litter size following intrauterine or cervical insemination of frozen/thawed semen with or without oxytocin administration. Theriogenology 62, 1236–1244.
ip t
Koracevic, D., Koracevic, G., Djordjevic, V., Andrejevic, S., Cosic, V., 2001. Method for the measurement of antioxidant activity in human fluids. J. Clin. Pathol. 54, 356-361.
cr
Larsen, L., Scheike, T., Jensen, T.K., Bonde, J.P., Ernst, E., Hjollund, N.H., 2000. Computer-assisted semen analysis parameters as predictors for fertility of men from the general population. The Danish
us
First Pregnancy Planner Study Team. Hum. Reprod. 15, 1562–1567.
Ma, H., Quan, F., Chen, D., Zheng, Y., Zhang, B., Wang, Y., Zhang, Y., 2011. Protective function of
an
alpha-lipoic acid on sperm motility and mitochondrial function during goat sperm-mediated gene transfer. Small Rumin. Res. 99, 191-198.
M
Maedler, K., Oberholzer, J., Bucher, P., Spinas, G.A., Donath, M.Y., 2003. Monounsaturated fatty acids prevent the deleterious effects of palmitate and high glucose on human pancreatic beta-cell turnover
d
and function. Diabetes 52, 726–33.
te
Marklund, S., Marklund, G., 1974. Involvement of superoxide anion radical in the auto-oxidation of pyragallol, and a convenient assay for superoxide dismutase. Europ. J. Biochem. 47, 469 – 474.
ce p
Marti, J.I., Marti, E., Cebrian-Perez, J.A., Muino-Blanco, T., 2003. Survival rate and antioxidant enzyme activity of ram spermatozoa after dilution with different extenders or selection by a dextran swim-up procedure. Theriogenology 60, 1025-1037.
Ac
Maxwell, W.M.C., Stojanov, T., 1996. Liquid storage of ram semen in the absence or presence of some antioxidants. Reprod. Fertil. Dev. 8, 1013–1020. Maxwell, W.M.C., Watson, P.F., 1996. Recent progress in the preservation of ram semen. Anim. Reprod. Sci. 42, 55–65. Menendez, J.A., Papadimitropoulou, A., Vellon, L., Lupu, R., 2006. A genomic explanation connecting "Mediterranean diet", olive oil and cancer: Oleic acid, the main monounsaturated Fatty acid of olive
20
Page 20 of 33
oil, induces formation of inhibitory "PEA3 transcription factor-PEA3 DNA binding site" complexes at the Her-2/neu (erbB-2) oncogene promoter in breast, ovarian and stomach cancer cells. Eur. J. Cancer 42, 2425-2432.
ip t
Michael, A., Alexopoulos, C., Pontiki, E., Hadjipavlou-Litina, D., Saratsis, P., Boscos, C., 2007. Effect of antioxidant supplementation on semen quality and reactive oxygen species of frozen-thawed canine
cr
spermatozoa. Theriogenology 68, 204-212.
Moradi, A.R., Malekinejad, H., Farrokhi-Ardabili, F., Bernousi, I., 2013. Royal Jelly improves the sperm
us
parameters of ram semen during liquid storage and serves as an antioxidant source. Small Rumin. Res. 113, 346-352.
an
Narang, D., Sood, S., Thomas, M., Dinda, A., Maulik, S., 2004. Effect of dietary palm olein oil on oxidative stress associated with ischemic–reperfusion injury in isolated rat heart. BMC Pharmacol. 4,
M
29.
Oudit, G., Sun, H., Kerfant, B., Crackower, M., Penninger, J., Backx, P., 2004. The role of
d
phosphoinositide-3 kinase and PTEN in cardiovascular physiology and disease. J. Mol. Cell. Cardiol.
te
37, 449-471.
Parthasarathy, S., Khoo, J.C., Miller, E., Barnett, J., Witztum, J. L., Steinberg, D., 1990. Low density
ce p
lipoprotein enriched in oleic acid is protected against oxidative modification: Implications for dietary prevention of atherosclerosis. Proc. Natl. Acad. Sci. USA 87, 3894-3898. Ruiz-Gutiérrez, V., Pérez-Espinosa, A., Vázquez, C.M., Santa-María. C., 1999. Effects of dietary fats
Ac
(fish, olive and high-oleic-acid sunflower oils) on lipid composition and antioxidant enzymes in rat liver. Br. J. Nut. 82, 233-241. Salamon, S., Maxwell, W.M.C., 2000. Storage of ram semen. Anim. Reprod. Sci. 62, 77-111. Sarıözkan, S., Bucak, M.N., Tuncer, P.B., Ulutas, P.A., Bilgen, A., 2009. The influence of cysteine and taurine on microscopic–oxidative stress parameters and fertilizing ability of bull semen following cryopreservation. Cryobiology 58, 134–138.
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Sheikholeslami Kandelousi, M.A., Arshami, J., Naserian, A.A., Abavisani, A., 2013. The Effect of addition omega-3, 6, 9, fatty acids on quality of bovine chilled and frozen-thawed sperms. Open Vet. J. 3, 47–52.
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Sicherle, C.C., Maia, M.S., Bicudo, S.D., Rodello, L., Azevedo, H.C., 2011. Lipid peroxidation and generation of hydrogen peroxide in frozen-thawed ram semen supplemented with catalase or Trolox.
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Small Rumin. Res. 95, 144–149.
Van Harken, D.R., Dixon, C.W., Heimberg, M., 1969. Hepatic Lipid Metabolism in Experimental
us
Diabetes. J. Biol. Chem. 224, 2278-2285. 385
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Figure legends
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Figure 1. Malondialdehyde (MDA) values (µmol/g protein; mean ± SE) in medium of rams following treatment with
389
different concentrations of oleic acid. A,B Values with different superscripts indicate difference (P < 0.05) among
390
groups at each time point. a,b,c,d Values with different superscripts indicate difference (P < 0.05) over time within the
391
experimental groups.
392
Figure 2. Malondialdehyde (MDA) values (µmol/g protein; mean ± SE) of ram spermatozoa following treatment
393
with different concentrations of oleic acid.
394
among groups at each time point.
395
within experimental groups.
396
Fig. 3. Total antioxidant capacity (TAC) (mmol/g protein; mean ± SE) in medium of rams following treatment with
397
different concentrations of oleic acid. A,B Values with different superscripts indicate significant difference (P < 0.05)
398
among groups at each time point.
399
within experimental groups.
400
Figure 4. Total antioxidant capacity (TAC) (mmol/g protein; mean ± SE) in spermatozoa of ram following treatment
401
with different concentrations of oleic acid.
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387
a,b
a,b,c
A,B
Values with different superscripts indicate difference (P < 0.05)
Values with different superscripts indicate a difference (P < 0.05) over time
Values with different superscripts indicate a difference (P < 0.05) over time
A,B
Values with different superscripts indicate differences (P < 0.05)
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Page 22 of 33
a,b,c,d
402
among groups at each time point.
403
within experimental groups.
404
Figure 5. Total amounts of nitric oxide (NO) (nmol/g protein; mean ± SE) in medium of rams following treatment
405
with different concentrations of oleic acid. There were no significant differences (P > 0.05) among groups at each
406
time point.
407
groups.
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Values with different superscripts indicate differences (P < 0.05) over time within experimental
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a,b,c
Values with different superscripts indicate differences (P < 0.05) over time
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408 409
Figure 6. Total amounts of nitric oxide (NO) (nmol/g protein; mean ± SE) in spermatozoa of ram following
410
treatment with different concentrations of oleic acid. A,B Values with different superscripts indicate differences (P <
411
0.05) among groups at each time point.
412
time within experimental groups.
413
Figure 7. Superoxide dismutase (SOD) activities (unit/mg protein; mean ± SE) in medium of rams following
414
treatment with different concentrations of oleic acid. There were no significant differences among groups at each
415
time point (P > 0.05).
416
experimental groups.
an
Values with different superscripts indicate differences (P < 0.05) over
M
a,b
d
Values with different superscripts indicate differences (P < 0.05) over time within
te
a,b
Figure 8. Superoxide dismutase (SOD) activities (unit/mg protein; mean ± SE) in spermatozoa of ram following
418
treatment with different concentrations of oleic acid. A,B Values with different superscripts indicate differences (P <
419
0.05) among groups at each time point.
420
time within experimental groups.
422
a,b
Values with different superscripts indicate differences (P < 0.05) over
Ac
421
ce p
417
423
23
Page 23 of 33
ip t
0.6
Control
Ad
cr
O 0.125
Bd Bd
0.4
Ac
Bd Bc
ABc ABc
Bc Bb
0.2
us
Ab Ab Ab Aa Ab Aa Aa Aa Aa Aa
0.0 Time 0h
O 0.25 O 0.5 O 1.0
an
Malondialdehyde (µmol/g protein)
Figure 1
Time 24h
Time 48h
Time 72h
te
d
M
Treatment Groups
ce p
1.0 0.8
Ac
Malondialdehyde (µmol/g protein)
Figure 2
Aa
0.6
Aa
Aa
Aa
Aa
Ab
ABaABa
Ab
Ab Ba Bab Ba
Ba Bab Ba
Control O 0.125
Ba Ba Bb Ba
O 0.25 O 0.5 O 1.0
0.4 0.2 0.0
Time 0h
Time 24h
Time 48h
Time 72h
Treatment Groups
Page 24 of 33
ip t
Figure 3
Ab
Aab Aa
O 0.125
Ab
BbcBbc Ba Bbc
Bc Ba Bc
Ab
1.5
Bc Ab
cr
2.0
Control
Aa Aa Aa Aa Aa
us
Ab
1.0 0.5 0.0 Time 0h
O 0.25 O 0.5 O 1.0
an
TAC (mmol/g protein)
2.5
Time 24h
Time 48h
Time 72h
te
d
M
Time of Experiment
4
2
Aa Aa Aa Aa Aa
ABb ABb Ab
Control O 0.125
Bb Bab Bbc Bbc ABc
Bb
O 0.25 Bd Bc Bb Bd
Abc
Ac
TAC (mmol/g protein)
6
ce p
Figure 4
O 0.5 O 1.0
Ac
0 Time 0h
Time 24h
Time 48h
Time 72h
Time of Experiment
Page 25 of 33
b
b c b
c b b
b
Control
b b
O 0.125
ab a b
O 0.25
ab
cr
ab
4
O 0.5
a a a
a
O 1.0
us
a
2
an
NO (nmol/g protein)
6
ip t
Figure 5
0 Time 0h
Time 24h
Time 48h
Time 72h
M
Treatment Groups
te
d
Figure 6
ce p
20
Ad
Aa
Aa
10
0
Aa
Aa
Aab Aa
Control O 0.125
Ac Bb Ba
Ab
Ba Ba Bb Bab
Ba Bb
O 0.25 O 0.5 O 1.0
Aa Aa Aa
Ac
NO (nmol/g protein)
30
Time 0h
Time 24h
Time 48h
Time 72h
Treatment Groups
Page 26 of 33
Control
30
O 0.125
a a a ab
20 a
a
a a b
b a
O 0.25
ab
O 0.5 O 1.0
a a a
cr
a
a ab
a
ip t
b
10
us
SOD activity (Unit/mg protein)
Figure 7
0 Time 0h
Time 24h
Time 48h
Time 72h
M
an
Treatment Groups
60
d te ce p
80
Aa Aa Aa Aa Aa
40 20 0
Ac
SOD activity (Unit/mg protein)
Figure 8
Time 0h
Control
Aab Aa Aa Aab Aa
Time 24h
Bb Bb Bb Ba
Bab Bab Bab Ba
O 0.125 O 0.25 O 0.5
Aab
O 1.0
Ab
Time 48h
Time 72h
Treatment Groups
Page 27 of 33
Table 4 Percentage of plasma membrane integrity test (HOST) of ram spermatozoa (mean ± SE) following enrichment with oleic acid and stored for various time points at 4 ◦C. Treatment
Time of storage (h) 24
48
Control
87.24 ± 1.79 Aa
76.68 ± 2.27 Ab
64.48 ± 2.00 Ac
O 0.125
87.55 ± 2.37 Aa
79.46 ± 2.32 Aa
68.79 ± 0.95 ABb
O 0.25
88.13 ± 1.84 Aa
82.50 ± 2.34 Aa
77.63 ± 2.39 BCab
70.43 ± 2.75 BCb
O 0.5
89.68 ± 2.25 Aa
83.39 ± 1.54 Aab
76.83 ± 1.81 BCbc
72.10 ± 2.63 BCc
O 1.0
88.77 ± 1.61 Aa
85.76 ± 1.63 Aab
78.73 ± 2.49 Cbc
us
cr
61.99 ± 1.51 ABb
74.53 ± 1.90 Cc
Values with different superscripts indicate differences (P < 0.05) among groups at each time point.
ce p
te
d
M
Values with different superscripts indicate significant differences (P < 0.05) between the data at the same row.
Ac
a,b,c
59.34 ± 2.12 Ac
an
A,B,C
72
ip t
0
Page 28 of 33
Table 3 Percentage of viable spermatozoa in ram semen (mean ± SE) following enrichment with oleic acid and stored for various time points at 4 ◦C. Treatment
Time of storage (h) 24
48
72
Control
95.32 ± 2.08 Aa
87.39 ± 3.71 Aab
79.77 ± 2.36 Abc
73.28 ± 1.60 Ac
O 0.125
95.26 ± 1.69 Aa
88.96 ± 2.81 Aab
81.03 ± 1.66 Abc
O 0.25
96.30 ± 1.30 Aa
91.87 ± 1.93 Ab
84.90 ± 2.75 Ac
O 0.5
93.23 ± 1.57 Aa
90.67 ± 1.67 Aa
85.28 ± 2.60 Ab
81.81 ± 2.29 Bb
O 1.0
94.12 ± 1.37 Aa
90.29 ± 1.70 Aab
85.78 ± 1.24 Ab
82.32 ± 2.36 Bb
78.76 ± 0.62 ABc
us
cr
80.76 ± 2.01 ABd
Values with different superscripts indicate difference (P < 0.05) among groups at each time point.
a,b,c,d
Values with different superscripts indicate significant differences (P < 0.05) between the data at the same
an
A,B
ip t
0
Ac
ce p
te
d
M
row.
Page 29 of 33
Treatment
Time of storage (h) 0
24
48
72
VAP (µm/s)
Control O 0.125 O 0.25 O 0.5 O 1.0
39.12 ± 1.23 Aa 38.48 ± 1.47 Aa 39.73 ± 1.31 Aa 37.75 ± 2.54 Aa 37.42 ± 1.43 Aa
25.47 ± 1.63 Ab 33.19 ± 1.77 Aab 31.47 ± 0.78 Ab 31.95 ± 2.70 Aab 30.20 ± 2.17 Aab
20.70 ± 0.94 Abc 27.07 ± 1.97 Abc 25.24 ± 0.66 Ac 24.64 ± 1.34 Ab 23.81 ± 1.97 Ab
18.63 ± 1.15 Ac 23.88 ± 2.24 Ac 23.34 ± 1.81 Ac 24.66 ± 1.65 Ab 24.23 ± 1.70 Ab
VSL (µm/s)
Control
25.43 ± 4.23 Aa
15.50 ± 1.29 Aab
13.57 ± 0.38 Ab
12.14 ± 1.13 Ab
O 0.125 O 0.25
Aa
23.92 ± 5.03 24.19 ± 5.23 Aa
21.46 ± 1.78 23.00 ± 1.22 Aa
Aa
16.19 ± 1.84 16.54 ± 1.62 Aa
14.79 ± 1.37 Aa 15.27 ± 1.22 Aa
O 0.5 O 1.0
22.80 ± 4.27 Aa 24.15 ± 1.42 Aa
20.71 ± 1.35 Aa 19.02 ± 1.97 Aab
17.01 ± 1.91 Aa 17.09 ± 1.93 Ab
15.71 ± 0.61 Aa 16.54 ± 0.85 Ab
Control
118.95 ± 3.87 Aa
93.57 ± 2.67 Ab
63.58 ± 3.66 Ac
55.30 ± 2.13 Ac
O 0.125 O 0.25 O 0.5
Aa
117.97 ± 2.08 114.77 ± 3.90 Aa 119.08 ± 1.07 Aa
O 1.0
123.00 ± 3.25 Aa
Control O 0.125
75.01 ± 1.04 Aa 76.38 ± 5.30 Aa
O 0.25 O 0.5 O 1.0
73.05 ± 1.44 Aa 76.99 ± 3.19 Aa 74.57 ± 4.71 Aa
Control O 0.125 O 0.25 O 0.5 O 1.0
LIN (%)
a,b,c
cr
an
64.51 ± 1.65 Bc 69.71 ± 2.38 BCb 71.56 ± 2.30 BCc
108.63 ± 3.79 Ba
83.15 ± 3.50 Bb
77.85 ± 2.52 Cb
63.41 ± 1.86 Ab 66.00 ± 3.03 Aa
67.82 ± 1.09 Ab 65.04 ± 2.96 Aa
65.86 ± 2.54 Ab 65.48 ± 2.61 Aa
67.37 ± 1.84 Aa 73.28 ± 1.87 Aa 70.11 ± 2.63 Aa
68.83 ± 3.15 Aa 72.40 ± 1.89 Aa 69.29 ± 1.38 Aa
67.06 ± 2.17 Aa 70.34 ± 2.12 Aa 70.77 ± 2.10 Aa
29.16 ± 0.86 Aa 29.94 ± 2.27 Aa
20.58 ± 1.03 Ab 24.17 ± 2.41 Aa
21.23 ± 1.37 Ab 24.56 ± 0.78 Aa
20.44 ± 1.41 Ab 23.28 ± 1.78 Aa
28.96 ± 5.87 Aa 28.65 ± 1.21 Aa 30.11 ± 1.87 Aa
23.88 ± 1.80 Aa 25.88 ± 1.57 Aa 25.36 ± 2.57 Aa
23.78 ± 4.57 Aa 26.37 ± 1.22 Aa 23.22 ± 1.76 Aa
23.21 ± 1.44 Aa 25.32 ± 1.79 Aa 24.58 ± 1.51 Aa
M
67.90 ± 2.76 75.57 ± 5.39 ABb 78.06 ± 1.95 Bc
d
ABc
Values with different superscripts indicate differences (P < 0.05) among groups at each time point.
Ac
A,B,C
100.50 ± 0.81 102.33 ± 1.87 ABa 106.68 ± 3.16 Bb
te
STR (%)
ABb
ce p
VCL (µm/s)
Aa
ip t
Parameter
us
Table 2 Spermatozoa motion parameters of ram (mean ± SE) assessed by CASA following enrichment with oleic acid and stored for various time points at 4 ◦C.
Values with different superscripts indicate significant differences (P < 0.05) among various time points in a group.
VCL: curvilinear velocity (µm/s), VSL: straight-line velocity (µm/s), VAP: average path velocity (µm/s), LIN: linearity ([VSL/VCL]×100, %), STR: straightness ([VSL/VAP]×100, %).
Page 30 of 33
Table 1 Percentage of total and forward progressive motility of spermatozoa (mean ± SE) in ram semen following enrichment with oleic acid and stored for various time points at 4 ◦C. Parameter
Treatment
Time of storage (h) 24
48
72
Control
93.61 ± 0.79 Aa
85.49 ± 1.58 Ab
77.50 ± 1.31 Ac
70.49 ± 1.83 Ad
O 0.125
91.89 ± 1.41 Aa
89.46 ± 1.56 Aa
80.56 ± 0.77 ABb
77.01 ± 1.20 Bb
O 0.25
93.20 ± 1.87 Aa
89.80 ± 0.98 Aa
85.51 ± 1.16 Bab
78.78 ± 1.73 Bb
O 0.5
94.19 ± 1.50 Aa
90.97 ± 1.40 Aab
84.60 ± 0.77 Bbc
80.93 ± 1.51 Bc
O 1.0
91.42 ± 1.40 Aa
88.09 ± 1.65 Aab
84.11 ± 1.73 Bab
81.58 ± 1.28 Bb
Forward progressive
Control
89.37 ± 1.18 Aa
58.29 ± 1.25 Ab
49.71 ± 1.28 Ac
43.77 ± 1.54 Ac
motility
O 0.125
88.64 ± 1.41 Aa
62.31 ± 1.22 ABb
56.33 ± 1.62 ABbc
52.37 ± 1.42 Bc
O 0.25
88.71 ± 2.20 Aa
63.35 ± 1.15 ABb
58.56 ± 1.86 Bbc
53.31 ± 1.13 Bc
O 0.5
87.75 ± 1.58 Aa
67.43 ± 2.11 Bb
61.89 ± 1.73 Bbc
56.53 ± 1.67 Bc
O 1.0
88.03 ± 1.88 Aa
68.46 ± 1.64 Bb
63.12 ± 1.79 Bbc
57.26 ± 2.13 Bc
cr
us
an
M
Values with different superscripts indicate difference (P < 0.05) among groups at each time point.
te
Values with different superscripts indicate significant differences (P < 0.05) between the data at the same row.
ce p
a,b,c,d
Ac
A,B
d
Total motility
ip t
0
Page 31 of 33
Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or
Ac
ce p
te
d
M
an
us
cr
ip t
organizations that could inappropriately influence or bias the content of the paper.
Page 32 of 33
Ram semen enrichment with oleic acid decreased the amounts of malondialdehyde and nitric oxide of spermatozoa during low temperature liquid storage. Moreover, it increased the total antioxidant capacity levels and superoxide dismutase activities of ram spermatozoa. Ultimately,
ip t
addition of oleic acid to ram semen enhanced the kinematics of spermatozoa during storage at
Ac
ce p
te
d
M
an
us
cr
refrigerator temperature.
Page 33 of 33