Increasing the Yield and Cryosurvival of Spermatozoa from Rhinoceros Ejaculates Using the Enzyme Papain
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Ethics Statement
2.3. Experimental Design
2.4. Animals and Semen Collection
2.5. Assessment of Initial Ejaculate and Sperm Membrane Characteristics
2.6. Treatment of Viscous Fractions with Papain and E-64
2.7. Dilution and Cryopreservation of Rhinoceros Spermatozoa
2.8. Thawing and Advanced In Vitro Semen Assessment
2.8.1. Computer-Assisted Sperm Analysis (CASA)
2.8.2. Flow Cytometric Analysis
2.9. Statistical Analysis
3. Results
3.1. Initial Ejaculate Characteristics Collected in Experiment 1 and 2 Prior to Freezing
3.2. Papain Improves the Quality of Spermatozoa Originating from Viscous, Low Sperm Fractions Both Prior to and after Cryopreservation
3.3. Papain Does Not Influence the Viability, Acrosome Integrity, Membrane Lipid Disorder, Intracellular ROS Production or DNA Integrity of Frozen-Thawed Rhinoceros Spermatozoa Assessed Using Flow Cytometry
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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Parameter | Ejaculate Fraction | |
---|---|---|
Sperm-Rich | Viscous, Low Sperm | |
Number of ejaculate fractions collected | 10 | 11 |
Volume (ml) | 2.6 ± 0.58 a | 15.1 ± 2.4 b |
Concentration (×106 spermatozoa/mL) | 863.3 ± 116.99 a | 236.0 ± 46.61 b |
Total sperm number per ejaculate (×109 spermatozoa) | 2.7 ± 0.93 | 3.4 ± 0.71 |
Time | Sperm Parameters | Viscous, Low Sperm Fraction | |
---|---|---|---|
Non-Treated Control | Papain-Treated | ||
Pre-freeze | Subjective motility (%) | 78.8 ± 2.39 a | 88.3 ± 1.7 b |
Post-thaw | TM (%) | 37.5 ± 10.56 a | 84.9 ± 1.60 b |
PM (%) | 25.4 ± 9.32 a | 56.6 ± 5.72 b | |
VAP | 43.8 ± 1.92 a | 74.4 ± 5.48 b | |
VCL | 77.1 ± 3.16 a | 115.8 ± 8.30 b | |
VSL | 34.7 ± 4.58 a | 57.6 ± 3.51 b | |
ALH | 3.7 ± 0.40 a | 4.5 ± 0.18 b | |
BCF | 39.6 ± 2.81 | 41.2 ± 0.23 | |
LIN | 50.8 ± 6.02 | 52.0 ± 1.86 | |
STR | 80.9 ± 5.92 | 78.3 ± 2.87 | |
Percent viable (%) Percent acrosome intact (%) Percent normal morphology (%) | 71.5 ± 3.66 a | 86.3 ± 3.33 b | |
68 ± 6.48 | 80.0 ± 3.70 | ||
70.8 ± 4.37 | 80.5 ± 2.72 |
Time Point | In Vitro Sperm Parameters | Sperm-Rich Control | Viscous Low Sperm Papain-Treated |
---|---|---|---|
Pre-freeze | Subjective motility (%) | 78.4 ± 6.66 | 76.6 ± 5.22 |
Percent viable (%) | 92.0 ± 1.10 | 93.2 ± 0.92 | |
Percent acrosome intact (%) | 88.2 ± 2.48 | 89.2 ± 1.86 | |
Percent normal morphology (%) | 82.2 ± 4.59 | 78.0 ± 5.10 | |
Post-thaw | TM (%) | 38.0 ± 5.50 a | 54.2 ± 6.13 b |
PM (%) | 21.8 ± 3.99 a | 40.9 ± 5.20 b | |
VAP (µm/s) | 48.3 ± 4.41 a | 68.8 ± 6.04 b | |
VCL (µm/s) | 78.0 ± 8.19 a | 101.2 ± 9.45 b | |
VSL (µm/s) | 37.9 ± 3.38 a | 58.9 ± 4.60 b | |
LIN (%) | 9.0 ± 2.33 a | 8.15 ± 1.98 b | |
STR (%) | 81.0 ± 2.04 a | 87.4 ± 1.79 b | |
BCF (%) | 39.0 ± 1.30 | 40.2 ± 0.96 | |
ALH (%) | 3.66 ± 0.31 | 3.57 ± 0.29 | |
Viable acrosome intact (%) | 40.2 ± 3.89 | 39.3 ± 1.90 | |
Membrane lipid disorder (median M540 fluor.) | 2966.4 ± 357.26 | 13,993.6 ± 4901.47 | |
Level of ROS (median H2DCFDA fluor.) | 17,278.9 ± 3923.57 | 9073.0 ± 2103.47 | |
DNA integrity (%) | 11.0 ± 0.71 | 8.51 ± 0.69 |
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Rickard, J.P.; Pool, K.; de Graaf, S.P.; Portas, T.; Rourke, N.; Wiesner, M.; Hildebrandt, T.B.; Göritz, F.; Hermes, R. Increasing the Yield and Cryosurvival of Spermatozoa from Rhinoceros Ejaculates Using the Enzyme Papain. Biology 2022, 11, 154. https://doi.org/10.3390/biology11020154
Rickard JP, Pool K, de Graaf SP, Portas T, Rourke N, Wiesner M, Hildebrandt TB, Göritz F, Hermes R. Increasing the Yield and Cryosurvival of Spermatozoa from Rhinoceros Ejaculates Using the Enzyme Papain. Biology. 2022; 11(2):154. https://doi.org/10.3390/biology11020154
Chicago/Turabian StyleRickard, Jessica P., Kelsey Pool, Simon P. de Graaf, Timothy Portas, Natalie Rourke, Miriam Wiesner, Thomas B. Hildebrandt, Frank Göritz, and Robert Hermes. 2022. "Increasing the Yield and Cryosurvival of Spermatozoa from Rhinoceros Ejaculates Using the Enzyme Papain" Biology 11, no. 2: 154. https://doi.org/10.3390/biology11020154