Supplementation of Thymoquinone Nanoparticles to Semen Extender Boosts Cryotolerance and Fertilizing Ability of Buffalo Bull Spermatozoa
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Ethical Statement
2.2. Preparation of Thymoquinone Nanoparticles (TQNPs)
2.3. Determination of Thymoquinone Nanoparticles Features
2.4. Animal Management and Semen Collection
2.5. Extender Preparation and Experimental Design
2.6. Freezing and Thawing
2.7. Assessments of Frozen–Thawed Semen Features
2.8. Computer-Assisted Sperm Analysis
2.9. Assessment of Acrosome Integrity
- -
- Viable sperm with an intact acrosome: The acrosomal region was stained in pink/purple, and the post-acrosomal region appeared white-stained. Viable sperm with exocytosed acrosome: post-acrosomal region was white, and acrosome was white (true acrosome exocytosis).
- -
- Non-viable sperm with an intact acrosome: the acrosomal region stained either dark pink or purple, and the post-acrosomal region stained either blue or dark blue.
- -
- Non-viable sperm with a reacted acrosome: the acrosomal region stained either in white or gray and the post-acrosomal region stained in blue (false acrosome exocytosis).
2.10. Assessment of Apoptosis through Flow Cytometry
- (a)
- Viable cells: without fluorescence signal and membrane dysfunction (A−/PI−).
- (b)
- Apoptotic sperm cells: viable cells labeled with Annexin V but without PI (A+/PI−).
- (c)
- Necrotic sperm cells: non-viable cells labeled with PI without Annexin V and with complete membrane loss (A−/PI+).
- (d)
- Necrotic sperm cells: Non-viable cells labeled with Annexin V and PI and with damaged permeable membranes (A+/PI+). Lastly, the number of spermatozoa in each previous category was recorded in each group.
2.11. Ultrastructure Assay of the Cryopreserved Spermatozoa
2.12. Oxidative Biomarkers Assays
2.13. Fertility Rate
2.14. Statistical Analysis
3. Results
3.1. Effects of TQNP on Buffalo Bull Semen after Equilibration (5 °C for 4 h)
3.2. Effects of TQNPs on Post-Thawed Buffalo Bull Semen
3.3. Effects of TQNPs on Kinematic Features of Post-Thawed Buffalo Bull Sperm
3.4. Effects of TQNPs on Acrosome Integrity of Post-Thawed Buffalo Bull Semen
3.5. Effects of TQNPs on Total Antioxidant Capacity and Oxidative and Nitrosative Biomarkers in Post-Thawed Buffalo Bull Semen
3.6. Effects of TQNPs on Apoptosis-like Changes
3.7. Sperm Ultrastructure
3.8. Effects of TQNPs on Fertility Trial
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Treatments 1 | Sperm Characteristics (%) | |||
---|---|---|---|---|
Progressive Motility | Viability | Membrane Integrity | Sperm Morphology | |
CON | 72.9 ± 1.01 c | 74.3 ± 1.36 c | 74.3 ± 0.81 b | 8.7 ± 0.47 a |
TQNP12.5 | 74.3 ± 1.30 bc | 76.1 ± 1.58 bc | 74.4 ± 0.95 b | 6.3 ± 0.68 b |
TQNP25 | 78.6 ± 1.43 ab | 80.1 ± 1.06 abc | 80.6 ± 1.17 a | 6.6 ± 0.37 b |
TQNP37.5 | 80.0 ± 1.54 a | 81.7 ± 1.76 ab | 80.3 ± 1.48 a | 6.4 ± 0.30 b |
TQNP50 | 80.7 ± 1.30 a | 82.4 ± 1.53 a | 81.6 ± 1.53 a | 7.1 ± 0.55 b |
p value | 0.0004 | 0.001 | <0.0001 | 0.009 |
Extender 1 | Sperm Characteristics (%) | |||
---|---|---|---|---|
Progressive Motility | Viability | Membrane Integrity | Sperm Morphology | |
CON | 41.4 ± 0.92 b | 43.7 ± 0.78 b | 44.1 ± 0.86 b | 9.7 ± 0.57 |
TQNP12.5 | 43.6 ± 0.92 ab | 45.6 ± 1.15 ab | 44.3 ± 1.32 b | 8.4 ± 0.65 |
TQNP25 | 47.1 ± 1.84 a | 48.0 ± 1.77 ab | 49.6 ± 2.20 a | 7.9 ± 0.34 |
TQNP37.5 | 47.9 ± 1.01 a | 48.7 ± 1.23 ab | 49.3 ± 1.08 a | 8.1 ± 0.46 |
TQNP50 | 48.6 ± 1.43 a | 49.7 ± 1.34 a | 50.3 ± 1.73 a | 9.3 ± 0.42 |
p value | 0.001 | 0.02 | 0.01 | 0.07 |
Variables (Unit) 1 | Treatments 2 | |||||
---|---|---|---|---|---|---|
CON | TQNP12.5 | TQNP25 | TQNP37.5 | TQNP50 | p Value | |
TM (%) | 54.5 ± 3.56 b | 57.6 ± 1.61 b | 57.4 ± 1.47 b | 57.6 ± 1.51 b | 67.6 ± 1.16 a | 0.003 |
PM (%) | 41.9 ± 1.98 d | 46.6 ± 1.21 cd | 51.4 ± 1.16 bc | 53.6 ± 0.93 b | 60.4 ± 0.98 a | <0.0001 |
DAP (µm) | 18.1 ± 0.40 c | 18.6 ± 0.46 c | 19.3 ± 0.50 bc | 22.5 ± 1.45 ab | 23.6 ± 0.68 a | <0.001 |
DCL (µm) | 28.3 ± 0.68 c | 29.2 ± 0.88 c | 31.0 ± 0.65 bc | 36.4 ± 2.48 ab | 37.6 ± 1.27 a | <0.001 |
DSL (µm) | 12.9 ± 0.27 b | 13.1 ± 0.56 b | 13.6 ± 0.42 b | 16.9 ± 1.11 a | 16.9 ± 0.49 a | <0.001 |
VAP (µm/s) | 41.0 ± 1.01 c | 42.1 ± 1.08 c | 44.0 ± 1.35 bc | 51.1 ± 3.29 ab | 53.5 ± 2.01 a | 0.0004 |
VCL (µm/s) | 63.8 ± 1.72 c | 65.5 ± 2.05 c | 70.4 ± 2.13 bc | 82.5 ± 5.64 ab | 85.0 ± 3.54 a | <0.001 |
VSL (µm/s) | 29.3 ± 0.69 b | 29.8 ± 1.11 b | 30.8 ± 1.17 b | 38.4 ± 2.50 a | 38.2 ± 1.43 a | <0.001 |
STR (%) | 71.0 ± 1.00 ab | 70.2 ± 1.74 ab | 69.6 ± 0.75 b | 74.2 ± 0.58 a | 70.8 ± 0.86 ab | 0.05 |
LIN (%) | 45.2 ± 0.49 | 45.2 ± 1.83 | 43.4 ± 0.68 | 46.0 ± 1.18 | 44.6 ± 0.51 | 0.50 |
WOB (%) | 64.0 ± 0.55 | 63.6 ± 1.29 | 62.0 ± 0.84 | 61.6 ± 1.29 | 62.6 ± 0.68 | 0.40 |
ALH (µm) | 2.6 ± 0.13 | 2.7 ± 0.14 | 2.7 ± 0.12 | 2.7 ± 0.16 | 2.9 ± 0.11 | 0.70 |
BCF (Hz) | 24.7 ± 0.80 abc | 24.0 ± 0.24 c | 24.4 ± 0.80 bc | 27.3 ± 0.88 ab | 27.5 ± 0.54 a | 0.003 |
Treatment 1 | Viable Sperm with Intact Acrosome (LSIA, %) | Viable Sperm with Exocytosed Acrosome (LSDA, %) | Non-Viable Sperm with an Intact Acrosome (DSIA, %) | Non-Viable Sperm with Exocytosed Acrosome (DSDA, %) |
---|---|---|---|---|
CON | 33.6 ± 1.08 c | 26.2 ± 2.73 a | 30.0 ± 2.07 | 10.2 ± 2.08 |
TQNP12.5 | 43.8 ± 0.97 b | 16.8 ± 1.36 b | 31.4 ± 1.36 | 8.0 ± 0.71 |
TQNP25 | 49.2 ± 0.97 a | 16.6 ± 0.51 b | 27.2 ± 0.58 | 7.0 ± 0.71 |
TQNP37.5 | 49.4 ± 0.93 a | 14.8 ± 0.66 b | 29.0 ± 1.30 | 6.8 ± 0.92 |
TQNP50 | 51.4 ± 1.21 a | 14.0 ± 0.55 b | 28.0 ± 1.52 | 6.6 ± 0.68 |
p value | <0.0001 | <0.0001 | 0.30 | 0.20 |
Treatment 1 | TAC (mM/L) | MDA (nmol/mL) | H2O2 (mM/L) | NO (µmol/L) |
---|---|---|---|---|
CON | 0.21 ± 0.03 c | 14.0 ± 0.91 a | 0.075 ± 0.004 a | 7.1 ± 0.35 |
TQNP12.5 | 0.25 ± 0.04 c | 13.0 ± 0.89 ab | 0.069 ± 0.006 a | 6.8 ± 0.54 |
TQNP25 | 0.39 ± 0.02 b | 10.6 ± 0.91 abc | 0.060 ± 0.002 ab | 6.5 ± 0.76 |
TQNP37.5 | 0.42 ± 0.03 ab | 10.2 ± 0.85 bc | 0.049 ± 0.004 bc | 5.7 ± 0.59 |
TQNP50 | 0.54 ± 0.03 a | 9.2 ± 0.78 c | 0.043 ± 0.003 c | 5.6 ± 0.31 |
p value | <0.0001 | 0.004 | <0.0001 | 0.20 |
Treatment 1 | Apoptosis-like Changes (%) | |||
---|---|---|---|---|
Viable (A−/PI−) | Early Apoptotic (A+/PI−) | Apoptotic (A+/PI+) | Necrotic (A−/PI+) | |
CON | 40.4 ± 0.38 d | 0.2 ± 0.02 c | 38.6 ± 1.07 a | 20.9 ± 0.79 a |
TQNP12.5 | 51.8 ± 1.13 c | 8.4 ± 0.50 a | 39.5 ± 0.67 a | 0.4 ± 0.03 d |
TQNP25 | 54.0 ± 1.37 c | 3.5 ± 0.85 b | 29.4 ± 2.18 b | 13.2 ± 1.75 b |
TQNP37.5 | 62.0 ± 0.55 b | 1.5 ± 0.00 bc | 30.7 ± 0.61 b | 5.8 ± 0.06 c |
TQNP50 | 67.4 ± 0.35 a | 2.8 ± 0.46 b | 29.5 ± 0.44 b | 0.3 ± 0.06 d |
p value | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
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Khalil, W.A.; Hassan, M.A.E.; El-Harairy, M.A.; Abdelnour, S.A. Supplementation of Thymoquinone Nanoparticles to Semen Extender Boosts Cryotolerance and Fertilizing Ability of Buffalo Bull Spermatozoa. Animals 2023, 13, 2973. https://doi.org/10.3390/ani13182973
Khalil WA, Hassan MAE, El-Harairy MA, Abdelnour SA. Supplementation of Thymoquinone Nanoparticles to Semen Extender Boosts Cryotolerance and Fertilizing Ability of Buffalo Bull Spermatozoa. Animals. 2023; 13(18):2973. https://doi.org/10.3390/ani13182973
Chicago/Turabian StyleKhalil, Wael A., Mahmoud A. E. Hassan, Mostafa A. El-Harairy, and Sameh A. Abdelnour. 2023. "Supplementation of Thymoquinone Nanoparticles to Semen Extender Boosts Cryotolerance and Fertilizing Ability of Buffalo Bull Spermatozoa" Animals 13, no. 18: 2973. https://doi.org/10.3390/ani13182973
APA StyleKhalil, W. A., Hassan, M. A. E., El-Harairy, M. A., & Abdelnour, S. A. (2023). Supplementation of Thymoquinone Nanoparticles to Semen Extender Boosts Cryotolerance and Fertilizing Ability of Buffalo Bull Spermatozoa. Animals, 13(18), 2973. https://doi.org/10.3390/ani13182973