Optimization of Sperm Management and Fertilization in Zebrafish (Danio rerio (Hamilton))
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Ethics Statement and Animals
2.2. Fish Culture and Stripping
2.3. Experimental Design
- (1)
- Testing spontaneous movement of spermatozoa.
- (2)
- Basic screening of four extenders from the literature with selections of the two most robust for further studies.
- (3)
- Detailed study of two extenders on how they are able to maintain motility and fertility depending on the storage period. Sperm motility, concentration, and seminal plasma osmolality were assayed using (a) milt from individual males, (b) pooled milt at time of collection, (c) individually collected milt and a storage pool of spermatozoa with good motility. In addition, visualization of the detailed movement of spermatozoa was performed using a high-speed video microscopy and a stroboscopic lamp. In the case of fertility, individually collected milt and a storage pool of spermatozoa with good motility were tested.
- (4)
- Finally, a suitable fertilization technique was specified.
2.4. First Step: Testing Spontaneous Movement of Spermatozoa
2.5. Second Step: Testing Extenders
2.6. Third Step: Testing of Sperm Storage, Spermatozoa Concentration, Seminal Plasma Osmolality, and Fertilization with Swim-Up Larvae Rates
2.6.1. Storage of Milt from Individual Males
2.6.2. Storage of Pooled Milt at Time of Collection
2.6.3. Individually Collected Milt and Storage Pool of Spermatozoa with Good Motility
2.6.4. Sperm Motility, Velocity, Concentration, and Osmolality of Seminal Plasma
2.6.5. Evaluation of Sperm Motility by Stroboscopic Illumination
2.6.6. Visualization of Motility of Sperm Flagella by High-Speed Video-Microscopy
2.6.7. Extender Evaluation by Fertilization, Swim-Up, and Malformation Larvae Rates
2.7. Fourth Step: Testing of Two Different Fertilization Methods
2.8. Statistical Analysis
3. Results
3.1. First Step: Testing “Spontaneous” Movement of Spermatozoa
3.2. Second Step: Testing Extenders
3.3. Third Step: Testing of Sperm Storage, Spermatozoa Concentration, Seminal Plasma Osmolality, and Fertilization with Swim-Up Larvae Rates
3.3.1. Storage Sperm from Individual Males
3.3.2. Storage of Pooled Milt at Time of Collection
3.3.3. Individually Collected Milt and Storage Pool of Spermatozoa with Good Motility
3.3.4. Sperm Concentration and Osmolality of Seminal Plasma
3.3.5. Visualization of Sperm Motility with Stroboscopic Light
3.3.6. Visualization of Sperm Motility in High-Speed Video Images
3.3.7. Extenders Evaluation by Fertilization and Swim-Up Larvae Rates
3.4. Fourth Step, Testing of the Two Different Fertilization Methods
4. Discussion
4.1. Sperm Motility
4.2. Milt Storage
4.3. Fertilization and Swim-Up Larvae Rates
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sperm Number per 80 eggs | Extenders | Volume of Sperm without Additional Extenders (μL) | Volume of Additional Extenders (μL) | Volume of Sperm with Additional Extenders (µL) | Volume of Hatchery Water (μL) |
---|---|---|---|---|---|
600,000 | E400 | 0.95 | 8.55 | 9.50 | 90.50 |
Kurokura | 1.00 | 9.00 | 10 | 90.00 | |
6,000,000 | E400 | 9.50 | 0 | 9.50 | 90.50 |
Kurokura | 10.10 | 0 | 10.10 | 89.90 |
Extenders | Without Activation | Motility (%) | |||
---|---|---|---|---|---|
(Hours after Activation with Distilled Water) | |||||
0 h | 6 h | 12 h | 24 h | ||
Control | 70% movement | 0.0 ± 0.0 dA | 0.0 ± 0.0 bA | 0.0 ± 0.0 bA | 0.0 ± 0.0 bA |
Kurokura | No movement | 80.0 ± 1.7 cA | 42.5 ±7.5 abB | 45.5 ± 3.5 aB | 1.2 ± 0.2 abC |
HBSS | <1% movement | 87.4 ± 0.5 abA | 15.4 ± 1.7 bB | 2.7 ± 0.8 bC | 0.0 ± 0.0 bC |
MHBSS | No movement | 89.0 ± 0.6 aA | 49.6 ± 7.0 aB | 30.3 ± 8.6 abBC | 6.8 ± 1.6 aC |
E400 | No movement | 82.5 ± 1.3 bcA | 65.0 ± 3.8 aB | 25.0 ± 5.0 abC | 0.0 ± 0.0 bD |
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Cheng, Y.; Franěk, R.; Rodina, M.; Xin, M.; Cosson, J.; Zhang, S.; Linhart, O. Optimization of Sperm Management and Fertilization in Zebrafish (Danio rerio (Hamilton)). Animals 2021, 11, 1558. https://doi.org/10.3390/ani11061558
Cheng Y, Franěk R, Rodina M, Xin M, Cosson J, Zhang S, Linhart O. Optimization of Sperm Management and Fertilization in Zebrafish (Danio rerio (Hamilton)). Animals. 2021; 11(6):1558. https://doi.org/10.3390/ani11061558
Chicago/Turabian StyleCheng, Yu, Roman Franěk, Marek Rodina, Miaomiao Xin, Jacky Cosson, Songpei Zhang, and Otomar Linhart. 2021. "Optimization of Sperm Management and Fertilization in Zebrafish (Danio rerio (Hamilton))" Animals 11, no. 6: 1558. https://doi.org/10.3390/ani11061558
APA StyleCheng, Y., Franěk, R., Rodina, M., Xin, M., Cosson, J., Zhang, S., & Linhart, O. (2021). Optimization of Sperm Management and Fertilization in Zebrafish (Danio rerio (Hamilton)). Animals, 11(6), 1558. https://doi.org/10.3390/ani11061558