Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology

Improving the effect of incubation and oxidative stress on thawed spermatozoa from red deer by using different antioxidant treatments

Álvaro E. Domínguez-Rebolledo A , María R. Fernández-Santos A B C , Alfonso Bisbal A , José Luis Ros-Santaella A , Manuel Ramón C , Manuel Carmona D , Felipe Martínez-Pastor E F G and J. Julián Garde A B F

A Biology of Reproduction Group, National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), 02071 Albacete, Spain.

B Institute for Regional Development (IDR), 02071 Albacete, Spain.

C Regional Center of Animal Selection and Reproduction (CERSYRA), JCCM, Valdepeñas, 13300 Ciudad Real, Spain.

D Cátedra de Química Agrícola, E.T.S.I. Agrónomos (UCLM), 02071 Albacete, Spain.

E ITRA-ULE, INDEGSAL, University of León, 24071 León, Spain.

F These authors contributed equally to this study.

G Corresponding author. Email:

Reproduction, Fertility and Development 22(5) 856-870
Submitted: 19 August 2009  Accepted: 10 December 2009   Published: 15 April 2010


Antioxidants could improve sperm media, extending the viability of spermatozoa and protecting their DNA. The protective ability of lipoic acid, melatonin, Trolox and crocin was tested on red deer spermatozoa incubated at 37°C. Cryopreserved spermatozoa were thawed and incubated with 1 mM or 0.1 mM of each antioxidant, with or without oxidative stress (100 μM Fe2+). Motility (CASA), viability, mitochondrial membrane potential and acrosomal status were assessed. Lipoperoxidation (malondialdehyde production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were checked at 4 h. Incubation alone increased ROS and decreased motility. Oxidative stress intensified these effects, increasing lipoperoxidation and DNA damage. Lipoic acid had little protective effect, whereas 1 mM melatonin showed limited protection. Trolox lowered ROS and lipoperoxidation both in oxidised and non-oxidised samples. In oxidised samples, Trolox prevented DNA and acrosomal damage, and ameliorated motility. Crocin at 1 mM showed similar results to Trolox, but noticeably stimulated motility and had no effect on lipoperoxidation. In a second experiment, a broader range of crocin and melatonin concentrations were tested, confirming the effects of crocin (positive effects noticeable at 0.5–0.75 mM), but showing an increase in lipoperoxidation at 2 mM. Melatonin was increasingly effective at 2.5 and 5 mM (ROS, lipoperoxidation and DNA status). Crocin seems a promising new antioxidant, but its particular effects on sperm physiology must be further studied, especially the consequences of motility stimulation and confirming its effect on lipoperoxidation. Melatonin might be useful at relatively high concentrations, compared to Trolox.

Additional keywords: crocin, lipoic acid, melatonin, Trolox.


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