Buck Semen Does Not Easily Succumb to Oxidative Stress

Semen processing and manipulation generally result in loss of sperm motility and sperm velocity due in part to oxidative stress. In this study we investigated the vulnerability of South African indigenous unimproved buck semen to oxidative stress induced by an oxidative stress inducing agent, namely, hydrogen peroxide (H2O2). Semen ejaculates were collected from four superior South African indigenous unimproved bucks in a total of ten collections and then each duplicate was treated with different concentrations of H2O2 in presence or absence of Dithiothreitol (DTT). Sperm motility and velocities were determined using the computer aided sperm class analyser (CASA). SYBR-14 and propidium iodide (PI) Live/Dead assay kit was used to determine cell viability and Yo-Pro-1 plus PI Apoptosis kit was used to determine apoptosis. Statistical analysis was performed on the data using SPSS version 17.0 for Windows (SPSS Inc., Chicago, IL). South African indigenous unimproved buck raw semen motility was between 97% with 98% viability and 0% apoptotic cells. Comparisons of the untreated controls at 0 and 3 hrs incubations revealed that after 3 hrs there was overall a decrease in the number viable cells with the majority of remaining cells exhibiting circular movements accompanied by high progressive (PM) and rapid (RAP) motilities. In treated South African indigenous unimproved buck semen, H2O2 marginally increased total motility (TM) with few apoptotic sperm cells while retaining high viability. Also, H2O2 increased straight line distances travelled of more than 4 fold as compared to untreated controls with no circularly moving cells. Moreover, inclusion of DTT, an antioxidant, had minimal effects on TM, RAP, curvilinear velocity (VCL), straight line velocity (VSL), linearity (LIN) How to cite this paper: Pilane, C.M., Bopape, M.A., Ntombizodwa, B. and Mapeka, M.H. (2019) Buck Semen Does Not Easily Succumb to Oxidative Stress. Open Journal of Animal Sciences, 9, 65-75. https://doi.org/10.4236/ojas.2019.91006 Received: November 6, 2018 Accepted: January 1, 2019 Published: January 4, 2019 Copyright © 2019 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access DOI: 10.4236/ojas.2019.91006 Jan. 4, 2019 65 Open Journal of Animal Sciences


Introduction
There is growing interest in the cryopreservation of the buck semen worldwide [1] [2].Cryopreservation is the technique of freezing cells and tissues at very low temperatures at which the biological material remains genetically stable but metabolically inert.In South Africa, cryopreservation of gametes from indigenous breeds has been recognised as a national priority due to their superior traits that are suitable for the local conditions [3] [4].In particular, the South African indigenous unimproved bucks are currently enjoying maximum attention as they have been previously ignored [5] [6].
Previously published data indicates that raw semen from this breed has TM above 83%, high PM, and high RAP accompanied by high viability [5].Accompanying these features were high VAP, LIN, STR and WOB, characteristic of superior quality semen [5].These experiments have also revealed that following cryopreservation, the South African indigenous unimproved buck semen showed decreased TM to less than 40% with low PM and RAP, accompanied by lower VAP, LIN, STR and WOB, indicating deterioration of semen quality [5].
Hydrogen peroxide (H 2 O 2 ) is often used as an experimental source of oxygen-derived free radicals such as superoxide radical (O 2− .)and hydroxyl radical ( .OH) which induces oxidative stress [7].Oxidative stress has been implicated as the main culprit for the decreased semen motility and semen velocity parameters in males leading to infertility [8].However, a cocktail of antioxidants can effectively improve semen parameters in infertile males [9].Other studies have indicated that the addition of antioxidants to cryopreservation medium can improve post-thaw sperm quality [10], while other antioxidants, mainly thiols, like DTT can prevent H 2 O 2 mediated loss of sperm motility [11].
Our previously published data indicated that in boars, the semen motility and semen velocity parameters were compromised under H 2 O 2 induced oxidative stress in the presence or absence of an anti-oxidant like DTT [12].Notably, TM, PM, RAP plus VAP, LIN, STR and WOB were all drastically decreased under oxidative stress, irrespective of the presence or absence of an antioxidants like DTT.The decreases in these parameters, accompanied by high rate of sperm cell Open Journal of Animal Sciences C. M. Pilane et al.
death via apoptosis and decreased viability, indicated that boar semen is highly susceptible to oxidative stress and DTT fail to rescue them [12].We have also shown recently that other antioxidants like α-tocopherol, taurine and cysteine do not restore boar semen motility and velocity parameters following oxidative stress associated with liquid preservation [13].Others have shown that post-thaw buck semen exhibited decreased DNA damage and lipid peroxidation accompanied by increased antioxidant enzyme activities with no changes in progressive motilities and sperm velocity parameters in the presence of antioxidants [14].
The South African unimproved buck semen has never been directly exposed to an oxidative stress inducing agent to test their susceptibility to oxidative stress.Hence, this study was conducted to assess whether the South African unimproved buck semen can easily succumb to oxidative stress induced by H 2 O 2 , in the presence or absence of an antioxidant like DTT.

Materials and Methods
The study was conducted at the Small Stock Research Unit of Agricultural Research Council, Germplasm Conservation & Reproductive Biotechnologies Unit, Irene, South Africa.The Agricultural Research Council-Irene campus is located at 25˚55'South; 28˚12'East.The institute is located in the Highveld region of South Africa and situated at an altitude of 1525 meters above sea level.Four superior South African indigenous bucks were stratified based on age (2 years) and weight (25 -45 kg).The South African indigenous unimproved bucks were in good health condition throughout the duration of the study.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Animals under the guidelines of the Agricultural Research Council, Animal Production Institute Animal Ethics Committee (APIEC2011/38).
Water was given ad libitum throughout the duration of the study.

Bucks Semen Collection and Processing
Semen was collected from four South African indigenous unimproved bucks using an electro-ejaculator, each with a total of ten collections.After collections, the semen samples were placed into the thermo-flask at 37˚C and transported to the laboratory.In the laboratory semen evaluations were performed within 1 h.Semen volume was measured by using the graduated falcon tube, pH was measured using the pH meter (Oaklon, EW35614-30, Cole-Parmer, USA), the sperm concentration was measured using the spectrophotometer (Jenway 6310 spectrophotometer, Bibby Scientific, England), the semen motility rates were assessed using the CASA system (Sperm Class Analyzer  [SCA] 5.0, Microptic, Barcelona, Spain), semen viability and apoptosis were evaluated using the SYBR/PI live/dead kit (Invitrogen, Molecular probes, USA) and Yo-Pro-1/PI apoptosis kit (Invitrogen, Molecular probes, USA) respectively.

Treatments
For semen treatment, H 2 O 2 stock solution was prepared in pre-warmed BO-Wash medium and kept at 4˚C until use.During the experiment, semen at equal concentration and volume was treated with pre-warmed H 2 O 2 stock to make 0, 5 µM, 50 µM and 200 µM concentrations in pre-warmed BO-Wash medium.The treated semen samples were then incubated at 37˚C for three hours in a humidified 5% CO 2 and 95% atmospheric air incubator (Sanyo, Japan).After three hours the samples were evaluated for total motility, progressive motility, rapid motility, semen velocities, sperm cell viability and sperm cell apoptosis.

Sperm Motility Rate
About 10 μl of raw semen were placed into 500 μl of BO-Wash medium in 15ml tube (Falcon  352099, USA).The tube was then kept in CO 2 incubator (Sanyo, Japan) adjusted to 39˚C.Five micro litres of semen was placed on the warm glass slide (~76 × 26 × 1 mm, Germany) and placed with a warmed cover slip (22 × 22 mm, Germany) over the microscope-warm plate (Omron) adjusted at 39˚C.The sperm motility rates were evaluated by computer assisted sperm analysis system (Sperm Class Analyzer  [SCA] 5.0, CASA, Microptic, Barcelona, Spain) at the 10× magnification (Nikon, China).The semen TM, PM, RAP and semen velocity parameters were then determined.

Viability Assay
For cell viability, SYBR-14 and PI Live/Dead kit was used and the cells were treated according to the manufacturer's recommendation (Invitrogen, Molecular probes, USA).Briefly, 50 µl of semen was diluted with pre-warmed BO-Wash to 1 ml and 5 µl of a 50 times diluted SYBR-14 was added to the cells followed by incubation at 37˚C for 10 minutes.After 10 minutes, 5 µl of PI was added to the cells followed by incubation for another 10 minutes.After 10 minutes 5 µl of cells was immediately placed on pre-warmed glass slide and observed under a fluorescent microscope at 10× magnifications (Olympus, model BX51).For this analysis, viable cells appeared green in colour while non-viable cells appeared red in colour.The percent cell viability was determined by counting the number of green cells out of three hundred cells in a field, divided by three.A correlation was then established between viability and H 2 O 2 treatment in the presence or absence of DTT.

Apoptosis Assay
To determine cell apoptosis, cells were treated with the Yo-Pro-1/PI staining kit solutions according to the manufacturer's recommendations (Invitrogen, Molecular probes, USA).In brief, cells were treated with 5 µl of Yo-Pro-1 and PI at

Data Analysis
Pearson's correlation coefficients were calculated to test the relationship between the motility and velocity rates parameters, namely, TM, PM, RAP, VCL, VSL, VAP, LIN, STR, WOB, apoptosis and viability against the treatments.Data were examined using the Kolmogorov-Smirnov test to determine their distribution, a multivariate analysis of variance was performed (ANOVA) and when significant differences were found, and the non-parametric Mann-Whitney U-test was used to compare pairs of values directly if data did not adjust to a normal distribution.All analyses were performed using SPSS version 17.0 for Windows (SPSS Inc., Chicago, IL).Significance was set at p < 0.05.

Results
The analysis of the sperm motility and velocity parameters, by definition, gives an excellent insight into the sperm quality profile (Table 1).The raw semen obtained from the South African indigenous unimproved bucks showed an average sperm concentration of 0.876 ± 321.55 × 10 9 cells/ml, with a pH of 7.5 ± 0.5, viability of 98 ± 1.5 and no apoptotic spermatozoa (Table 2).The analysed sperm quality profile of this raw semen revealed the percentage TM value of97.0 ± 3.95, PM of 29.3 ± 1.3 and RAP of 8.37 ± 8.01 accompanied by VCL of 70.3 ± 1.56, VSL of 30.83 ± 4.32, VAP of 44.0 ± 7.21 and LIN plus STR of 64.6 ± 5.32 and 67.73 ± 5.28, respectively (Table 3).
The analysis of sperm cell viability using the SYBR-14/PI revealed a clear distinction between the viable and non-viable cells, with viable cells fluorescing Table 1.Definitions of the sperm motility and velocity parameters.green due to SYBR-14 absorption and non-viable cells absorbing SYBR-14 and PI and appearing as dominant red fluorescence (Figure 1).The class distribution of sperm cells according to average speed and distance travelled indicated untreated controls at 0 hrs and 3 hrs revealed a decrease in the number of motile cells as the viability decreased (Figure 2).At 0 hrs the RAP and PM motilities appears to be dominant however, after 3 hrs, NPM become dominant with more cells moving in circles or semi-circles (Figure 2) This class distribution of sperm cells according to average speed and distance travelled also revealed that while the H 2 O 2 treatment decreased the overall number of viable cells, the progressive and rapid motilities remained high accompanied by the elimination of circular moving cells as compared to untreated controls (Figure 3).After 3 hrs of incubation, DTT, H 2 O 2 and H 2 O 2 plus DTT appeared to activate sperm cells to travel longer distances in excess of 100 µm as compared to untreated controls (Figure 3).
Interestingly, all H 2 O 2 concentrations used increased TM, but 50 µM H 2 O 2 increased both TM and RAP in the presence of DTT (Figure 3, Table 4).
The untreated control semen had TM = 50.0± 33.Values with different subscripts with the same column differ significantly p ≤ 0.05).
while the other semen velocity parameters of treated semen were not significantly different from their corresponding untreated controls (Table 3).In the DOI: 10.4236/ojas.2019.91006

Conclusion
South African indigenous unimproved buck semen characterisation is currently been addressed and knowledge of their vulnerability to oxidative stress is necessary.Interestingly South African unimproved buck semen shows robust resistance to ROS effects, as shown by improved or unaffected motility and velocity parameters.Currently, in livestock, semen motility parameters are being used as a measure of their fertility potential.In South African unimproved buck semen, fertilizing potential might not be an issue since they show resistance to ROS, however the use of antioxidant should still be considered.While these data show buck semen resistance to ROS, more research is required to investigate how and why buck semen has resistance to ROS as compared to semen from boars.
the same time and incubated for 10 minutes.After incubation, 5 µl of the stained cells was placed on a slide and viewed under a fluorescent microscope at 60× magnification (Olympus, model BX51).Four populations of cells were obtained, the light green or clear (live cells), the dark green (apoptotic cells), and red (dead/necrotic cells) and the red plus green cells (dead cells).The percent cell C. M. Pilane et al. apoptotic cells were determined by counting the number of dark green cells out of three hundred cells in a field.A correlation was then established between the apoptosis and H 2 O 2 treatment in the presence or absence of DTT.
Percent of sperm showing any movement % Progressive Motility (PM) Percent of sperm moving rapidly and in a straight path % Rapid Motility (RAP) Percent of sperm travelling at a speed of 25 µm/sec or faster % Curvilinear Velocity (VCL) Time-average velocity of sperm head along its actual path µm/s Straight Line Velocity (VSL) Time-average velocity of sperm head projected along straight line µmlength to total length of curvilinear trajectory; LIN = VSL/VCL % Straightness (STR) Ratio of projected length to average velocity of sperm head along a spatial trajectory, STR = VSL/VAP % Wobble (WOB) Expression of the degree of oscillation of the curvilinear path about its spatial average path; WOB = VAP/VCL %

Figure 1 .
Figure 1.(A) The SYBR-14/PI cell viability staining of buck semen where red fluorescence represent non-viable sperm cells and green fluorescence represent viable sperm cells (10× magnification); (B) The Yo-Pro-1/PI apoptosis staining of buck semen showing apoptotic sperm cells in green fluorescence, moribund/dying sperm cells with dual fluorescence, dead sperm cells in red fluorescence while live cells had clear/light green fluorescence (60× magnification).

Figure 2 .
Figure 2. The class distribution of sperm cells according to their average speed showing the rapidly moving spermatozoa (RAP, red), Progressive motility (PM, green), Non-progressive motility (NPM, blue) and static (yellow) for untreated controls at (A) 0 hrs and (B) 3 hrs, bar = 25 µm.

Table 2 .
South African indigenous unimproved buck raw semen viability, apoptosis and macroscopic evaluations represented as mean ± SD.

Table 3 .
The motility and velocity rates of the South African indigenous unimproved buck raw semen represented as mean ± SD.

Table 4 .
The sperm motility and velocity rates of treated South African indigenous unimproved buck semen represented as mean ± SD.