Enhanced sperm cryopreservation using zinc- and copper-doped hydroxyapatite with glutathione and raffinose: effects on motility, viability, deoxyribonucleic acid integrity, and oxidative stress markers

Abstract

Objective: To study the protective effects of zinc- and copper-doped hydroxyapatite (H) combined with glutathione (GSH) and raffinose (R) on mouse sperm motility, viability, deoxyribonucleic acid (DNA) integrity, and oxidative stress markers before and after cryopreservation.

Design: Experimental laboratory-controlled study evaluating the efficacy of combined cryoprotective formulations during sperm cryopreservation.

Subjects: Sperm samples collected from 10 healthy adult albino mice were used for in vitro cryopreservation experiments.

Intervention: Samples were divided into six groups: a control group using a commercial cryopreservation medium and five experimental groups supplemented with H, GSH, and R, either alone or in combination (H+GSH+R). All samples were cryopreserved at -196°C for 72 hours and subsequently thawed for analysis.

Main outcome measures: Postthaw sperm motility, viability, and DNA fragmentation were evaluated, along with biochemical markers of oxidative stress, including total antioxidant capacity, total oxidant status, lipid peroxidation, protein oxidation, and enzymatic activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase.

Results: The H+GSH+R group demonstrated significantly higher motility and lower DNA fragmentation and mortality compared with the control. Total antioxidant capacity increased prefreezing, although total oxidant status was markedly reduced postthaw. Lipid and protein oxidation decreased significantly, and antioxidant enzyme activities were enhanced in the H+GSH+R group.

Conclusion: Zinc- and copper-doped H combined with GSH and R effectively improves sperm cryosurvival by enhancing antioxidant defenses and reducing oxidative damage. This formulation provides a promising cryoprotective strategy for optimizing assisted reproductive technologies.

Keywords: Cryopreservation media; DNA integrity; Zn- and Cu-doped hydroxyapatite; oxidative stress; sperm.