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. 2024 Aug 25;14(17):2465.
doi: 10.3390/ani14172465.

Stallion Sperm Freezing with Different Extenders: Role of Antioxidant Activity and Nitric Oxide Production

Raffaele Boni  1 , Raffaella Ruggiero  1 , Tommaso Di Palma  2 , Maria Antonietta Ferrara  3 , Graziano Preziosi  3 , Stefano Cecchini Gualandi  1
Affiliations

Stallion Sperm Freezing with Different Extenders: Role of Antioxidant Activity and Nitric Oxide Production

Raffaele Boni et al. Animals (Basel). .

Abstract

Sensitivity to freezing remains a critical issue in stallion semen cryopreservation procedures. To explore this topic in-depth, semen was collected from ten stallions, diluted with three different extenders, transported to the laboratory, and then centrifuged and frozen with four different extenders. We conducted analyses of sperm kinetics, mitochondrial membrane potential (MMP), and hydrogen peroxide content both before and after freezing. Additionally, we assessed antioxidant activity using the ABTS and FRAP methods and measured nitric oxide stable metabolites (NOx) in the blank extenders, seminal plasma, and extenders conditioned by spermatozoa before and after freezing. We found significant variability in the antioxidant activity and NOx content of the blank extenders and the seminal plasma. In the seminal plasma, ABTS-based antioxidant activity and NOx values were correlated with some sperm kinematic parameters and MMP in refrigerated semen, while no correlation was observed in frozen sperm parameters. Sperm function varied significantly between stallions but not between extenders, either before or after freezing. However, significant differences in antioxidant activities and NOx values were found among extenders conditioned following freezing. These results provide new insights into the factors contributing to the variability in individual stallions' tolerance to sperm freezing.

Keywords: conditioned extenders; equine; freezing injuries; nitric oxide stable metabolites; sperm quality; total antioxidant capacity.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Regression lines and correlation coefficients (R) between refrigerated and frozen/thawed (storage) sperm including the effects of the individual stallions and the extenders for sperm kinetics–sperm total (TotMot) and progressive (Prog) motility, curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP)–mitochondrial membrane potential (MMP), and hydrogen peroxide content (ROS) as well as antioxidant activity (ABTS and FRAP) and nitric oxide stable metabolites (NOx) assessed in the conditioned extenders. The percentages of total (TotMot) and progressive (Prog) sperm motility have been expressed in arcsine.
Figure 1
Figure 1
Regression lines and correlation coefficients (R) between refrigerated and frozen/thawed (storage) sperm including the effects of the individual stallions and the extenders for sperm kinetics–sperm total (TotMot) and progressive (Prog) motility, curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP)–mitochondrial membrane potential (MMP), and hydrogen peroxide content (ROS) as well as antioxidant activity (ABTS and FRAP) and nitric oxide stable metabolites (NOx) assessed in the conditioned extenders. The percentages of total (TotMot) and progressive (Prog) sperm motility have been expressed in arcsine.
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