The present study was conducted to assess boar sperm susceptibility to oxidative stress generated by hydrogen peroxide (H 2O 2). Semen was collected in replicates from three experimental large white boars using the gloved-hand technique. Semen ejaculates from three boars were treated with different concentrations of H 2O 2 for three hours. SYBR-14 and Propidium Iodide (PI) Live/ Dead assay kit was used to determine cell viability, and Yo-pro-1 and PI apoptosis kit was used to determine cell death, namely, apoptosis. Boar sperm motility obtained using computer aided sperm analysis (CASA) was between 90% and 100% with more than 98% viability with 0% apoptotic cells. In H 2O 2 treated boar sperm cells, rapid (RAP) and progressive motility (PM) increased. Also, H 2O 2 treatment induced a high positive correlation with apoptosis but high negative correlation with viability. Hydrogen peroxide decreased boar semen total motility (TM) by 10%. In addition, most of the boar sperm cells became apoptotic and lost 55% of viability under oxidative stress induced by H2O2. This study illustrated that boar semen was more susceptible to oxidative stress induced by H 2O 2.
Semen quality is an important factor in the livestock industry for successful quality breeding and cryopreservation [
Reactive oxygen species with relatively long half-life inhibits human sperm motility at high concentrations [
Currently, computerized analyzing systems, like computer aided sperm class analyzer (CASA), provide the best way for semen motility assessment of collected semen [
The study was conducted at the Pig 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. Three exotic Large White boars were used for this study at the ages of 2 to 3 years of age. The Large White boars 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 (APIEC/13/002). Boars were fed grower diet and water was given ad libitum throughout the duration of the study.
Semen samples were collected in spring (September-November) from the experimental boars. From each of the three superior boars used in this study, six ejaculates were collected separately using the gloved-hand technique in a 300 mL glass beaker. The filtered semen fraction was sealed with a gauze filter inside a pre-warmed (39˚C) insulated thermos flask. After collection, the semen samples were placed into the thermo-flask at 39˚C and transported to the laboratory where semen evaluations were performed within 1 h of collection. For all the replicates, semen volume was measured by using the graduated falcon tube, pH was measured using the litmus paper, the sperm concentration was measured using the spectrophotometer (Jenway 6310 spectrophotometer, Bibby Scientific, England), the sperm motility rates were assessed using the CASA system (Sperm Class Analyzer® [SCA] 5.0, Microptic, Barcelona, Spain), sperm 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 under a fluorescent microscope (Olympus model, BX-51).
For semen treatment, H2O2 stock solution was prepared in pre-warmed BO-Wash and kept at 4˚C until use. During the experiment, semen at equal concentration and volume was treated with pre-warmed H2O2 stock to make 0, 5 µM, 50 µM and 200 µM concentrations. The treated cells were then incubated at 37˚C for three hours in a humidified 5% CO2 and 95% atmospheric air incubator (Sanyo, Japan). After three hours the cells were evaluated for total motility, progressive motility, rapid motility, semen velocities, viability and apoptosis.
The sperm concentration was determined with the aid of spectrophotometer using the following formula (76) × [21.39 × (Absorbance) − 1.09] for boar sperm concentration determinations. Briefly, 3 mL of a 2.9% sodium citrate was placed in a cuvette and placed in a spectrophotometer set at 650 nm wavelength before calibration. After calibration, the absorbance of the diluted semen sample was recorded and the given formula was used to determine the semen concentration.
Briefly, 10 μl of raw semen was placed into 500 μL of BO-Wash medium in 15 mL tube (Falcon® 352099, USA). The tube was then kept in CO2 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 (
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 propidium iodide was added to the cells followed by incubation for an additional 10 minutes. After 10 minutes, 5 µL of cells were immediately placed on pre-warmed glass slide and observed under a fluorescent microscope (Olympus, model BX51). For this analysis, viable
Parameter | Definition | Unit |
---|---|---|
Total Motility (TM) | 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 traveling 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 | µm/s |
Average Path Velocity (VAP) | Time-average velocity of sperm head projected along its spatial trajectory | µm/s |
Linearity (LIN) | Ratio of projected length 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 | % |
cells appeared green in colour due to SYBR while non-viable cells appear red due to propidium iodide. The percent cell viability was determined by counting the number of green cells out of a hundred cells in a field. The data was represented in a table as percent viability versus H2O2 concentrations.
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 the same time and incubated for 10 minutes. After incubation, 5 µL of the stained cells was placed on a pre-warmed slide and viewed under a fluorescent microscope (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 and green cells (dead cells). The percent cell apoptotic cells were determined by counting the number of dark green cells out of a hundred cells in a field. The data was represented in a table as percent apoptosis versus H2O2 concentrations.
The apoptosis and viability data was expressed as a percentage of total cells observed in a field, out of 300 sperm cells observed. Pearson’s correlation coefficients were calculated to test the relationship between the motility rates parameters (
Our results indicates that the SYBR-14/PI staining of sperm cells for viability can clearly distinguish between viable (green) and non-viable/dead cells (red) (
Raw semen | Concentration (1 × 109 sperm cells/mL) | pH | Volume (mL) | Viability (%) | Apoptosis (%) |
---|---|---|---|---|---|
Boar | 0.987 ± 186.21a | 7.0 ± 0.0a | 144 ± 16.3b | 97.5 ± 2.5a | 0.00a |
a, bValues with different subscripts with the same column differ significantly (P £ 0.05).
Raw semen | TM% | PM% | RAP% | VCL (µm/s) | VSL (µm/s) | VAP (µm/s) | LIN% | STR% | WOB% |
---|---|---|---|---|---|---|---|---|---|
Boar | 94.07 ± 4.11a | 23.3 ± 4.94a | 4.0 ± 0.93a | 66.2 ± 3.69a | 28.4 ± 2.52a | 48.27 ± 2.89a | 58.67 ± 2.81a | 73.3 ± 7.72a | 77.95 ± 4.95a |
a, bValues with different subscripts with the same column differ significantly (P £ 0.05).
Treatment of semen from the boars with hydrogen peroxide for 3 hours, in the presence or absence of DTT, revealed that TM in boars was decreased by 5 µM, 50 µM and 200 µM hydrogen peroxide from 41.7 ± 12.6 to 29.1 ± 5.2, 36.4 ± 13.7 and 31.5 ± 10.1, respectively and the presence of DTT had no effect. For the PM of boar semen, hydrogen peroxide had no effect but DTT improved PM in 200 µM hydrogen peroxide treated semen from 2.63 ± 1.09 to 22.7 ± 3.12. Hydrogen peroxide also increased the RAP of boar semen in a concentration dependent manner and DTT had no effect. Also, this hydrogen peroxide concentration significantly decreased LIN, (from 50.5 ± 7.89 to 36.3 ± 8.07) and STR (from 67.23 ± 4.57 to 50.0 ± 7.89) but had no significant effect on other semen velocity parameters (
Apoptosis analysis of hydrogen peroxide treated boar semen revealed an increase in apoptosis in the presence or absence of DTT. This data is illustrated by Pearson’s correlation coefficient analysis (
Boar Semen | % TM | %PM | %RAP | VCL (µm/s) | VSL (µm/s) | VAP (µm/s) | %LIN | %STR | %WOB |
---|---|---|---|---|---|---|---|---|---|
Control | 41.7 ± 12.6a | 3.3 ± 1.4a | 0.7 ± 0.78a | 46.03 ± 15a | 24.27 ± 12a | 36.00 ± 16.6a | 50.5 ± 7.89a | 67.23 ± 4.57a | 79.4 ± 4.94a |
5 µM H2O2 | 29.1 ± 5.2a | 1.0 ± 0.4a | 0.2 ± 0.21a | 59.1 ± 2.01a | 27.67 ± 6.9a | 47.83 ± 3.86a | 47.0 ± 12.2a | 57.43 ± 11.3a | 80.96 ± 5.63a |
50 µM H2O2 | 36.4 ± 13.7a | 2.3 ± 0.4a | 0.83 ± 0.40a | 61.57 ± 4.7a | 26.7 ± 7.58a | 46.63 ± 11.6a | 43.50 ± 12.9a | 57.13 ± 10.7a | 75.2 ± 14.9a |
200 µM H2O2 | 31.5 ± 10.1a | 2.6 ± 1.0a | 1.067 ± 0.6a | 66.7 ± 5.76a | 24.83 ± 7.1a | 49.3 ± 12.81a | 36.60 ± 8.07a | 50.0 ± 1.74a | 72.7 ± 13.89a |
DTT | 45.4 ± 16.4a | 7.2 ± 4.8b | 0.93 ± 0.4a | 61.3 ± 16.9a | 21.3 ± 7.26a | 44.67 ± 13.7a | 35.1 ± 6.63a | 47.3 ± 1.51a | 75.0 ± 11.57a |
5 µM H2O2 + DTT | 29.1 ± 17a | 2.5 ± 1.3a | 0.3 ± 0.22a | 66.6 ± 4.18a | 24.4 ± 5.53a | 47.6 ± 10.30a | 36.4 ± 7.32a | 51.3 ± 5.50a | 70.9 ± 11.5a |
50 µM H2O2 + DTT | 36.4 ± 13.7a | 3.6 ± 3.2a | 1.63 ± 1.33a | 70.2 ± 6.24a | 23.5 ± 8.71a | 51.07 ± 11.6a | 33.1 ± 11.98a | 44.53 ± 11.1a | 72.1 ± 11.63a |
200 µM H2O2 + DTT | 37.4 ± 24.9a | 22.7 ± 3.1c | 0.63 ± 0.09a | 35.5 ± 5.16a | 53.4 ± 5.0b | 60.07 ± 3.91b | 53.47 ± 5.09a | 64.17 ± 3.51a | 67.55 ± 4.10a |
a, b, cValues with different subscripts with the same column differ significantly (P £ 0.05).
−DTT | +DTT | |
---|---|---|
Boar Semen | Boar Semen | |
Total Motility | −0.368a | −0.00844b |
Rapid Motility | 0.7988a | −0.532b |
Progressive Motility | 0.0753a | 0.9268b |
VCL | −0.743a | 0.252b |
VSL | −0.352a | 0.997b |
VAP | 0.623a | 0.978a |
LIN | 0.256a | 0.975b |
STR | −0.839a | 0.733b |
WOB | −0.934a | −0.849a |
Apoptosis | 0.919a | 0.9841a |
Viability | −0.9996a | −0.0958b |
a, bValues with different subscripts with the same row differ significantly (P £ 0.05).
This study indicates that hydrogen peroxide induces oxidative damage to semen which is accompanied by changes in the semen velocity parameters, semen motility parameters and semen viability, and these are also appears to be species dependent. In boars hydrogen peroxide had variable effects on TM in the presence or absence or DTT. In boars, hydrogen peroxide decreased TM in the presence or absence of DTT. In a similar study, Griveau et al. [
Also, 200 µM hydrogen peroxide decreased VAP, LIN and STR but had no effect on VCL. The antioxidant, DTT, decreased VCL and WOB while LIN and STR which restored to raw semen values while VAP and VSL were improved. In addition, treatment with 200 µM hydrogen peroxide, DTT had a decreasing effect on VCL. The decrease in semen VCL is due to a reduction in both the angle of lateral head displacement (ALH) and the beat cross frequency (BCF). A decrease in ALH means the sperm head is moving less from side to side while a decrease in BCF means these actions occur slower. Ironically, VCL in boars semen treated with 50 µM hydrogen peroxide is improved by DTT, indicating that ROS levels must be lower in boar semen for antioxidants like DTT to take effect. Aitken et al. [
In boars, hydrogen peroxide could not improve VAP, but the presence of DTT enhanced VAP by only 10%. Hydrogen peroxide decreased LIN, but LIN was restored to raw semen values by DTT. Also, STR and WOB were decreased by hydrogen peroxide and further decreased by the presence of DTT compared to raw semen values. This indicates that ROS can compromise boar sperm velocity parameters which cannot be rescued by an antioxidant, like DTT. Together, this datum demonstrates that the use of antioxidants in any boar semen processing is essential during cryopreservation.
The authors wish to acknowledge Nedambale TL for intellectual support, Agricultural Research Council (ARC) and National Research Foundation (NRF) for providing necessary facilities and financial support.
Cyril Mpho Pilane,Malebogo Audrey Bopape,Mohleko Helen Mapeka,Thivhilaheli Richard Netshirovha, (2016) Assessment of the Susceptibility of Boar Semen to Oxidative Stress. Open Journal of Animal Sciences,06,123-130. doi: 10.4236/ojas.2016.62015