Optimization of Vitrification Protocols for Feline Epididymal Spermatozoa: Impact on Post-Warming Sperm Quality and Fertilizing Potential
Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Animals and Reagents
2.2. Experimental Design
2.2.1. Experiment 1: Effect of Sucrose Concentration on Post-Warming Sperm Quality
2.2.2. Experiment 2: Effect of Disaccharide Type (Sucrose vs. Trehalose) on Post-Warming Sperm Quality
2.2.3. Experiment 3: Effects of Stepwise Mixing Volume and Equilibration Time on Post-Warming Sperm Quality
2.2.4. Experiment 4: Assessment of Fertilizing Potential Following Optimized Vitrification Protocol
2.3. Feline Epididymal Sperm Collection
2.4. Vitrification and Warming
2.5. Sperm Quality Evaluation
2.6. In Vitro Production of Feline Embryos
2.7. Assessment of Embryo Developmental Rate and Quality
2.8. Statistical Analysis
3. Results
3.1. Experiment 1: Effect of Sucrose Concentration on Quality of Vitrified Warmed Feline Epididymal Sperm
3.2. Experiment 2: Effect of Disaccharide Type (Sucrose vs. Trehalose) on Quality of Vitrified Warmed Feline Epididymal Sperm
3.3. Experiment 3: Effects of Stepwise Mixing Volume and Equilibration Time on Quality of Vitrified Warmed Feline Epididymal Sperm
3.4. Experiment 4: Effects of Optimized Vitrification Protocol on Fertilizing Potential of Vitrified Warmed Feline Epididymal Sperm
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
ARTs | Assisted Reproductive Technologies |
BSA | Bovine Serum Albumin |
COC | Cumulus–Oocyte Complex |
DAPI | 4′,6-Diamidino-2-Phenylindole |
DMSO | Dimethyl Sulfoxide |
EthD-1 | Ethidium Homodimer-1 |
FITC-PNA | Fluorescein Isothiocyanate–Peanut Agglutinin |
HM | Holding Medium |
ICSI | Intracytoplasmic Sperm Injection |
IVF | In Vitro Fertilization |
IVM | In Vitro Maturation |
LN2 | Liquid Nitrogen |
PBS | Phosphate-Buffered Saline |
ROS | Reactive Oxygen Species |
SEM | Standard Error of the Mean |
SOF | Synthetic Oviductal Fluid |
TUNEL | Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling |
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Solution | Pre-Vitrified Motility (%) | Post-Warming Motility (%) | Pre-Vitrified Viability (%) | Post-Warming Viability (%) |
---|---|---|---|---|
Sucrose 0.25 M | 80.0 ± 0 a | 5.0 ± 0 b | 95.63 ± 1.19 a | 6.38 ± 1.19 b |
Sucrose 0.5 M | 80.0 ± 0 a | 10.0 ± 0 b | 95.63 ± 1.19 a | 13.88 ± 2.47 b |
Sucrose 0.75 M | 62.5 ± 5 a | 2.5 ± 2.89 b | 95.63 ± 1.19 a | 6.25 ± 1.91 b |
Solution | Pre-Vitrified Motility (%) | Post-Warming Motility (%) | Pre-Vitrified Viability (%) | Post-Warming Viability (%) |
---|---|---|---|---|
Sucrose 0.5 M | 80.0 ± 0 a | 8.33 ± 2.58 b | 96.12 ± 1.19 a | 15.95 ± 4.95 b |
Trehalose 0.5 M | 80.0 ± 0 a | 10.0 ± 0 b | 96.12 ± 1.19 a | 18.92 ± 5.70 b |
Stepwise Mixing Volume | Equilibration Time | Pre- Vitrified Motility (%) | Post-Warming Motility (%) | Pre- Vitrified Viability (%) | Post-Warming Viability (%) |
---|---|---|---|---|---|
5 µL drop | 0 min | 80.0 ± 0 a | 8.75 ± 4.79 b | 96.38 ± 1.49 a | 14.5 ± 7.85 b |
10 min | 80.0 ± 0 a | 10.0 ± 4.08 b | 96.3 ± 2.56 a | 14.13 ± 7.20 b | |
10 µL drop | 0 min | 80.0 ± 0 a | 12.5 ± 2.89 b | 95.08 ± 1.52 a | 19.73 ± 9.40 b |
10 min | 80.0 ± 0 a | 11.25 ± 2.5 b | 96.3 ± 2.56 a | 19.95 ± 8.90 b | |
20 µL drop | 0 min | 82.5 ± 5 a | 21.25 ± 6.29 b | 96.75 ± 1.64 a | 32.15 ± 8.42 b |
10 min | 80.0 ± 0 a | 13.75 ± 2.5 b | 96.3 ± 2.56 a | 22.3 ± 8.66 b |
Sperm | Intact (%) | Partial Damage (%) | Complete Loss (%) |
---|---|---|---|
Fresh (Control) | 84.81 ± 4.22 a | 9.31 ± 2.22 a | 4.29 ± 4.01 a |
Vitrified (Treatment) | 22.86 ± 3.33 b | 65.04 ± 1.64 b | 12.10 ± 2.02 a |
Sperm | Total Oocyte (N) | Cleavage (%) | Blastocyst (%) * | Total Blastocyst Cell Number |
---|---|---|---|---|
Fresh (Control) | 51 | 61.55 ± 11.41 a | 51.03 ± 12.91 a | 540.88 ± 289.43 a |
Vitrified (Treatment) | 69 | 68.35 ± 14.16 a | 33.97 ± 9.21 a | 522.28 ± 335.31 a |
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Kallayanathum, W.; Udomthanaisit, L.; Tharasanit, T. Optimization of Vitrification Protocols for Feline Epididymal Spermatozoa: Impact on Post-Warming Sperm Quality and Fertilizing Potential. Animals 2025, 15, 1919. https://doi.org/10.3390/ani15131919
Kallayanathum W, Udomthanaisit L, Tharasanit T. Optimization of Vitrification Protocols for Feline Epididymal Spermatozoa: Impact on Post-Warming Sperm Quality and Fertilizing Potential. Animals. 2025; 15(13):1919. https://doi.org/10.3390/ani15131919
Chicago/Turabian StyleKallayanathum, Wirakan, Larindhorn Udomthanaisit, and Theerawat Tharasanit. 2025. "Optimization of Vitrification Protocols for Feline Epididymal Spermatozoa: Impact on Post-Warming Sperm Quality and Fertilizing Potential" Animals 15, no. 13: 1919. https://doi.org/10.3390/ani15131919
APA StyleKallayanathum, W., Udomthanaisit, L., & Tharasanit, T. (2025). Optimization of Vitrification Protocols for Feline Epididymal Spermatozoa: Impact on Post-Warming Sperm Quality and Fertilizing Potential. Animals, 15(13), 1919. https://doi.org/10.3390/ani15131919