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Cholestanol-loaded-cyclodextrin improves the quality of stallion spermatozoa after cryopreservation
E A Moraes1, W C G Matos1, J K Graham2
1Department of Animal Science, Federal University of San Francisco Valley, Petrolina, PE, 56304205, Brazil.
Insights
Adding cholesterol or cholestanol loaded cyclodextrins to stallion sperm before cryopreservation significantly improved post-thaw motility and viability. Cholestanol loaded cyclodextrins also enhanced sperm binding efficiency after cryopreservation.
Area of Science:
- Reproductive Biology
- Cryobiology
- Animal Science
Background:
- Cryopreservation of stallion sperm is crucial for genetic preservation.
- Optimizing cryosurvival rates remains a challenge in equine reproduction.
- Lipid supplementation strategies are being explored to enhance sperm cryotolerance.
Purpose of the Study:
- To evaluate the efficacy of cholesterol loaded cyclodextrins (CLC) and cholestanol loaded cyclodextrins (CnLC) in improving stallion sperm cryosurvival.
- To compare the effects of different concentrations of CLC and CnLC on post-thaw sperm motility, viability, and function.
- To assess the impact of these additives on sperm binding to the chicken egg perivitelline membrane (CEPM) post-cryopreservation.
Main Methods:
- Stallion ejaculates were diluted and treated with varying concentrations of CLC or CnLC.
- Treated sperm were incubated to allow lipid incorporation before cryopreservation.
- Sperm motility, viability, and CEPM binding were assessed post-thaw using standard laboratory techniques.
Main Results:
- Both CLC and CnLC treatments significantly increased post-thaw motility and viability compared to controls (P<0.05).
- CnLC addition resulted in a higher number of sperm binding to CEPM post-cryopreservation compared to controls (P<0.05).
- Higher percentages of motile and viable sperm were achieved with CLC and CnLC treatments.
Conclusions:
- Cholesterol loaded cyclodextrins and cholestanol loaded cyclodextrins enhance post-thaw motility and viability of equine sperm.
- CnLC offers superior sperm binding efficiency to CEPM after cryopreservation.
- These findings suggest that lipid-loaded cyclodextrins are promising additives for improving stallion sperm cryopreservation protocols.
Abstract:
This study was to compare the effect of adding cholesterol or cholestanol loaded cyclodextrins in stallion sperm prior to cryopreservation to optimize sperm cryosurvival. Ejaculates from each of eight stallions were diluted to 120 million cells in a S-MEDIUM diluent. The diluted sperm were sub-divided into three treatments: no additive (control); 0.75mg of cyclodextrin pre-loaded with cholesterol (CLC)/120 million sperm (positive control); 1.5mg CLC/120 million sperm; 0.75mg of cyclodextrin pre-loaded with cholestanol (CnLC)/120 million sperm; and 1.5mg CnLC/120 million sperm. To set the experiments, the treated sperm were incubated for 15min at 22°C to allow for the incorporation of cholesterol or cholestanol. In each experiment, treated sperm incubated for 15min at 22°C to allow for incorporation of cholesterol or cholestanol. The samples were then diluted 1:5 (v/v) with Lactose-Egg Yolk diluent and cooled to 5°C over a 2h period. Loaded into 0.25ml polyvinylchloride straws, frozen in liquid nitrogen vapor for 10min, and then plunged into liquid nitrogen until further use. Higher percentages of motile sperm and viable cells were achieved after thawing for stallion sperm treated with CLC and CnLC compared to control (P<0.05). Addition of CnLC also resulted in more number sperm binding to chicken egg perivitelline membrane (CEPM) after cryopreservation than cholestanol and control sperm (P<0.05). In conclusion, CnLC and CLC improved the percentage of post-thaw motility of equine sperm and CnLC provided greater binding efficiency.
