This study was conducted to investigate the effects of different levels of α-tocopherol in cryopreservation media on the bull sperm quality after thawing. Semen samples were collected from six Holstein bulls using artificial vagina twice a week. Semen samples were pooled to eliminate individual differences and then divided into four equal parts for freezing with extenders containing different concentrations of α-tocopherol according to experimental groups as follows: 0 (control), 1.2 mM (E1), 2.4 mM (E2) and 4.8 mM (E4). Motion characteristics, viability, plasma membrane functionality, lipid peroxidation and H
2
O
2
status were determined after thawing. Results showed that malondialdehyde (MDA) concentration was significantly lower in E4 (6.1±0.6 nmol/mL) than E1 and E2 extenders (8.1±0.6 nmol/mL and 8±0.6 nmol/mL, respectively). Also, the lowest significant concentration of H
2
O
2
was observed in E4 (3.2±0.13 nmol/mL) compared to E1 (4 ±0.1 nmol/mL), E2 (5.3± 0.1 nmol/mL) and control (6.7±0.1 nmol/mL). Moreover, E2 and E4 produced the highest significant motility (74.2±1.6%, 75.9±1.6%), viability (78.2±1.8%, 76.1±1.8%) and membrane functionality (73±1.6%, 70.5±1.6%) compared to other groups. It can be concluded that α-tocopherol at the concentration of 4.8 mM can be an efficient antioxidant additive in Bioxcell extender for cryopreservation of bull semen.
Additional key words:antioxidantbioxcelllipidperoxidationsperm
H
2
O
2Abbreviations used:LPO (lipid peroxidation)MDA (malondialdehyde)ROS (reactive oxygen species)The authors received no specific funding for this work.
Author´s contributions:
Conceived and designed the experiments; performed the experiments: FM, AP, and LS. Analysed the data; contributed reagents/materials/analysis tools: FM, AP, DC, and LS. Wrote the paper: FM, AP, DC, AC and LS.
Competing interests:
The authors have declared that no competing interests exist.
Introduction
Improvement in livestock production techniques, especially artificial insemination and semen freezing are the leading causes of accelerated rate of genetic selection (
Barbas & Mascarenhas, 2009
;
Forouzanfar
et al
., 2010
). Semen cryopreservation has allowed specific opportunities for conservation of genetic resources through sperm banks, guarantee of a constant commercial supply of semen, and collaboration in breed improvement programs by artificial insemination (
Bucak
et al
., 2007
;
Masoudi
et al
., 2017
). Semen cryopreservation may lead to oxidative, chemical and physical damages on sperm membrane leading to reduction of sperm viability and fertility (
Evans
et al
., 1987
;
Watson, 2000
;
Emamverdi
et al
., 2014
;
Najafi
et al
., 2014a
;
Sariözkan
et al
., 2015
). Freezing process mostly leads to loss of motility, acrosomal and plasma membrane functionality of spermatozoa (
Tuncer
et al
., 2011
;
Najafi
et al
., 2013
;
Shahverdi
et al
., 2015
). Moreover, because of high amount of polyunsaturated fatty acids in mammalian spermatozoa (plasma membrane), bull spermatozoa is very susceptible to oxidative stress which can influence on the quality and fertility potential of spermatozoa (
Zanganeh
et al
., 2013
). Antioxidant enzymes such as glutathione peroxidase and superoxide dismutase have crucial roles to maintain defense mechanisms against oxidative stress-induced damages in semen (
Tuncer
et al
., 2010
). However, antioxidant capacity in spermatozoa may be insufficient to prevent oxidative stress during the freeze–thawing process (
Ernster
et al
., 1992
;
Najafi
et al
., 2014b
;
Masoudi
et al
., 2016a
). Therefore, addition of suitable antioxidants to the extenders is suggested to reduce oxidative damages during freeze–thawing of bull spermatozoa (
Büyükleblebici
et al
., 2014
).
Alpha-tocopherol is a well-known lipid peroxidation inhibitor in biological membranes, acting as a scavenger of reactive oxygen species (ROS), preventing oxidative damage during cryopreservation of bull semen (
O’Flaherty
et al
., 1997
). A water-soluble vitamin E analogue (Trolox) improved sperm motility and mitochondrial membrane functionality during liquid storage of boar semen (
Cerolini
et al
., 2000
). In a study by
Dalvit
et al
. (1998)
, vitamin E increased the rate of fertilization when added to the bull semen extender. Alpha-tocopherol also improved total motility and viability of bull spermatozoa after freeze-thawing (
Nasiri
et al
., 2012
). This observation may be related to the protective effects of α-tocopherol against ROS (
Towhidi & Parks, 2012
). Moreover, dietary supplementation of vitamin E has been reported to increase reproductive capacity in chicken (
Khan
et al
., 2012
), boar (
Brzezinska-Slebodzinska
et al
., 1995
), rabbit (
Yousef, 2010
), ram (
Masoudi
et al
., 2016b
) and buck (
Majid
et al
., 2015
).
Although there are several reports for beneficial effects of α-tocopherol on cryopreservation of bull spermatozoa, there is no report for consideration of cellular parameters such as H
2
O
2
and sperm parameters after thawing. Therefore, the present study was conducted to determine the potential effects of different concentrations of α-tocopherol in Bioxcell extender for cryopreservation of bull semen. H
2
O
2
concentration and lipid peroxidation were assessed as an ROS scavenging in sperm cells. Moreover, sperm parameters, including motion characteristics, viability, membrane functionality and morphology were also evaluated after freeze-thawing-thawing.
Material and methods
The chemicals used in this study were purchased from Sigma (St. Louis, MO, USA), and Merck (Darmstadt, Germany), unless otherwise indicated.
Animal and semen collection
Six fertile Holstein bulls (aged 3-6 years) under uniform conditions with feeding based on National Research Council (
NRC, 2001
) were used in this study. Semen samples were collected using artificial vagina from six bulls twice a week during three weeks (in six replicates). Immediately after collection, semen volume and sperm concentration, sperm motility and morphology were evaluated. Semen samples were accepted for experiment if the following criteria were met: volume of 5-10 mL, concentration of >1×10
9
spermatozoa/mL, total motility > 70%, and abnormal morphology < 10%. Then, ejaculates from bulls in each day were pooled and divided into four groups according to experimental treatments.
Antioxidant treatments, semen dilution and cryopreservation
Alpha-tocopherol was added to the Bioxcell (IMV Technologies, L’Agile, France) to yield four different final concentrations: 0 (control), 1.2 mM (E1), 2.4 mM (E2) and 4.8 mM (E4). Because the α-tocopherol was not soluble in water, ethanol 0.05% was used to solve it before addition to extender. Pooled semen was divided into four equal aliquots and diluted with four extenders containing different concentrations of α-tocopherol to a final concentration of approximately 60×10
6
spermatozoa/mL (15×10
6
total spermatozoa in each 0.25 mL straw). Diluted semen samples were cooled to 4°C for 4 h. Subsequently semen was frozen at a programmed rate of -3°C /min from +4 to -10°C; -40°C/min from -10 to -100°C; and -20°C/min from -100 to -140°C in a digital freezing machine (Digitcool 5300 ZB 250, IMV, France). Thereafter, the straws were plunged into liquid nitrogen for storage. For sperm evaluation, straws were thawed individually at 37°C for 30 s in a water bath. Sperm evaluation was performed on all semen samples immediately after thawing.
Motility and velocity parameters
Motion characteristics of rewarmed sperm were measured using a computer-assisted sperm analysis (CASA, CEROS vers. 12.3; Hamilton-Thorne Bio-sciences, Beverly, MA, USA). Sperm sample was placed in a chamber (38
o
C, Leja 4; 20 mm height; Leja Products, Luzernestraat B.V., Holland) and then loaded chamber placed on the warm stage of the microscope (37°C). Afterwards, three randomly selected microscopic fields were examined. Motility data was characterized as follows: total motility (MOT,%); progressive motility (PROG,%); average path velocity (VAP, µm/s); straight line velocity (VSL, µm/s); curvilinear velocity (VCL, µm/s); amplitude of lateral head displacement (ALH, µm); straightness (STR,%); linearity (LIN,%). At least 200 spermatozoa were assessed in each CASA analysis.
Membrane functionality (HOST)
Hypo-osmotic swelling test (HOST) was designed to determine membrane functionality as described by
Revell & Mrode (1994)
, with a slight modification. Briefly, 10 µL of semen was mixed with 100 µL of a hypo-osmotic solution [100 mOsm/L, 57.6 mM fructose and 19.2 mM sodium citrate] in a 1.5 mL test tube and incubated at 37
o
C for 30 min. After incubation, the mixture was homogenized and evaluated under a phase-contrast microscope (CKX41, Olympus, Tokyo, Japan). A total of 200 spermatozoa were counted in at least five different microscopic fields at ×400. The percentage of spermatozoa with swollen and curved tails was recorded.
Sperm viability
Viability was assessed using Eosin–Nigrosine staining method (
Najafi
et al
., 2013
). Briefly, 20 µL aliquot from sperm suspension was stained by 20 µl Eosin–Nigrosine dye. Then, smears were prepared on a warm slide and spread the stain with a second slide. Twenty hundred sperm were counted under phase-contrast at 1000 × magnification. Sperm displaying partial or complete purple staining were considered nonviable; only sperm showing strict exclusion of stain were counted as viable. The viability was assessed by counting 200 spermatozoa under phase-contrast at ×1000 (CKX41, Olympus, Tokyo, Japan).
Malondialdehyde concentration assay
MDA as an index of lipid peroxidation was measured according to the method described by
Esterbauer & Cheeseman (1990)
. Briefly, 1 mL of sperm suspension (250×10
6
spermatozoa/mL) was mixed with 1 mL of cold trichloroacetic acid (20% w/v). The precipitate was pelleted by centrifuging (963×
g
for 15 min), and 1 mL of the supernatant was removed and incubated with 1 mL of thiobarbituric acid (0.67% w/v) in a boiling water bath at 100°C for 10 min. After cooling, the absorbance was determined by a spectrophotometer (UV-1200, Shimadzu, Japan) at 532 nm.
Measurement of hydrogen peroxide
The concentration of H
2
O
2
in thawed sperm and extenders were measured by the Phenol Red colorimetric method described by
da Silva Maia
et al
. (2010)
. Briefly, a sample of 100 µL of thawed sperm containing approximately 40×10
6
spermatozoa was incubated at 37 °C for 30 min in 1.0 mL of buffered phenol red solution. After incubation, the samples were centrifuged at 2000×g for 10 min, and the supernatant was decanted into a microtube. Then, 10 µL of NaOH solution was added to supernatant. The same procedure was used to determine the concentration of H
2
O
2
generated in the extender. The assay was performed in duplicate, and the absorbance of the samples was read at 610 nm, at 25°C, in a UV–vis, double beam spectrophotometer (Lambda 25, Perkin Elmer, Beaconsfield, UK). The concentration of H
2
O
2
in the sample was determined by comparing the absorbance obtained with a standard curve.
Statistical analysis
All data were checked for normal distribution by Shapiro–Wilk test and analyzed using Proc GLM of SAS 9.1 (SAS Inst, Cary, NC, USA). Six replicates were used for evaluation. Statistical differences among various group means were determined by Tukey’s test and the values of
p
<0.05 were considered to be statistically significant. Results are shown as mean±SEM.
Results
Table 1
shows the percentage of motion parameters in the frozen-thawed bull semen in extenders supplemented with different concentrations of α-tocopherol. Total motility and progressive motility were significantly higher in E4 (75.9±1.6%, 43.3±1.3%) and E2 (74.2±1.6%, 39.1±1.3%) compared to control (61.3±1.6%, 30.5±1.3%), respectively. No significant difference was detected for E0 and E1 for total motility and progressive motility.
Effect of different extenders on post-thawed bull spermatozoa motility and motion parameters (mean±SEM).
Data related to the viability, membrane functionality, MDA concentration and H
2
O
2
are presented in the
Table 2
.
The higher significant viability in frozen-thawed sperm was observed in E2 and E4 (78.2±1.8%, 76.1±1.8%, respectively) compared to control (61.3 ±1.8%). Moreover, E2 and E4 produced higher significant membrane functionality (73±1.6%, 70.5 ±1.6%, respectively) compared to control (60.5±1.6%). No significant difference was detected for control and E1 for viability and membrane functionality.
Viability and membrane functionality (HOST) in bull spermatozoa diluted in different extenders (mean±SEM).
For MDA concentration, although E4 produced the lowest significant concentration of MDA (6.1±0.6 nmol/mL) compared to E1 and E2 (8.1±0.6 nmol/mL and 8.0±0.6 nmol/mL, respectively), no significant difference was observed between E4 and control (7.2±0.6 nmol/mL). Moreover, the lowest significant concentration of H
2
O
2
was observed in E4 (3.2±0.1 nmol/mL) compared to E2 (4.0±0.1 nmol/mL), E1 (5.3± 0.1 nmol/mL) and control (6.7± 0.1 nmol/mL). The difference between E2 and E1 was also significant compared to control.
Discussion
Mammalian spermatozoa contains high amount of polyunsaturated fatty acids in plasma membrane which makes them susceptible to oxidative stress, especially during freeze-thaw process (
Purdy, 2006
;
Tuncer
et al
., 2011
). Damages to membrane matrix causes destruction of structural and biochemical organs of sperm leading to reduction of sperm motility and fertility (
Watson, 1976
). Frozen-thawed bull spermatozoa is more proxidized than fresh sperm due to lose of intracellular antioxidant capacity in sperm (
Tuncer
et al
., 2010
). Therefore, optimization of bull spermatozoa freezing procedure using an antioxidant additive can be an efficient strategy to improve the quality of post-thawed sperm. In this study, 2.4 and 4.8 mM α-tocopherol showed a suitable protective effect against freezing damages. However, for H
2
O
2
, extender supplemented with 4.8 mM α-tocopherol produced better results compared to 2.4 mM. It was clear that ROS accumulated during the cooling, equilibration, freeze-thawing and post-thaw incubation of sperm. Our results showed that a- tocopherol has suitable cryo-protective effects through its ability to quench ROS accumulation, which is in agreement with several studies that stated analogous of vitamin E in extenders increased the recovery rate of motility and viability of sperm (
Donoghue & Donoghue, 1997
;
Surai
et al
., 1998
;
Silva
et al
., 2013
). Moreover, similar to our study,
Domínguez-Rebolledo
et al
. (2010)
reported that motion characteristics and acrosomal integrity of epididymal red deer spermatozoa were improved when Trolox was added to incubation medium after thawing. However, some studies do not confirm these results because analogues of vitamin E had negative effects when it was added to refrigeration medium of ram (
Mata-Campuzano
et al
., 2014
) and red deer (
Anel-López
et al
., 2012
) spermatozoa. This discrepancy may be related to selected dose of vitamin E, dilution rate or preservation procedure.
One of the main roles of α-tocopherol in cryopreservation media is reduction of MDA and consequently improvement in sperm motility (
Suleiman
et al
., 1996
). Sperm is highly susceptible to lipid peroxidation (LPO). The spontaneous membrane LPO disrupts the structure of membrane via ROS (
Bucak
et al
., 2008
) which ultimately lead to loss of sperm function, such as reduction in membrane functionality, sperm motility and fertility potential (
Bansal & Bilaspuri, 2011
). Alpha-tocopherol is believed to be the primary component of the antioxidant system of spermatozoa, and is regarded as one of the major membrane protectants against ROS and LPO (
Bansal & Bilaspuri, 2009
). In the present study, we found that exposure of sperm to the α-tocopherol resulted in less H
2
O
2
during the cryopreservation process. These results are in agreement with
Amini
et al
. (2015)
and
Martínez-Páramo
et al
. (2012)
, who obtained lower lipid peroxidation of sperm in response to α-tocopherol in rooster and sea bass, respectively, after cryopreservation. Moreover, reduction in amount of H
2
O
2
in this experiment in response to α-tocopherol may be due to potential of α-tocopherol to the phenoxyl radicals stabilization. Similar results have also been reported by
Breininger
et al
. (2005)
, who stated that α-tocopherol suppressed the ROS in boar spermatozoa after thawing.
Using α-tocopherol for reduction of MDA was our interest to evaluate the measurement of MDA in spermatozoa cells and their effects on the sperm performance. Alpha tocopherol can also increase the electron transfer during oxidative stress resulting to stable phenoxyl radical (
Davies
et al
., 1988
). However, results of MDA in this study were not as might be expected, because we thought a reduction in MDA after supplementation of our extender with α-tocopherol would happen. This behavior may be due to the connection of α-tocopherol because antioxidants may influence on the MDA in the low concentration (
Zhandi & Sharafi, 2015
).
Taking together, we tested the effects of α-tocopherol in the wide range (0-4.8 mM) for cryopreservation of bull spermatozoa. The higher results were obtained in high doses of α-tocopherol (2.4-4.8 mM). It should be noted that the efficiency of antioxidants are affected by various factors such as component of buffer, cryoprotectants and incubation time which resulted to obtain different outcomes in literatures. Cryoprotectants such as egg yolk and soybean or milk have different antioxidant capacity (
Alvarez-Rodríguez
et al
., 2013
). It is possible that component of Bioxcell have some effects on the optimum level of α-tocopherol. Finally, we understand that addition of optimum dose of α-tocopherol could improve bull sperm quality indices.
In summary, our findings demonstrate that concentrations of α-tocopherol (2.4-4.8 mM) in Bioxcell can be efficient for preservation of bull spermatozoa in freezing status, although this issue must be tested in fertility trials.
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