The effects of crocin on frozen-thawed rooster sperm parameters

Articles in Press

Document Type : Research Article

Authors

Soroush Entezari ¹

Vahid Hamedianasl ²

Pezhman Mirshokraei ³

¹ Graduated with a DVM from Ferdowsi University of Mashhad, Mashhad, Iran.

² Department of Basic Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.

³ Department of Clinical Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.

10.22067/ijvst.2026.98404.1654

Abstract

This study investigated the effect of crocin on the post-thaw sperm-quality parameters of frozen rooster sperm. In this study, eight Ross 308 roosters were used to collect semen samples. After the initial evaluation, the semen was diluted using a laboratory-made diluent that closely resembled Beltsville Poultry Semen Extender (BPSE). Crocin was evaluated at 0, 0.5, 1, 2, and 4 mM in Stage 1. Because the 4 mM group showed undesirable sperm morphology and the lowest viability, only this concentration was excluded from the Stage 2 follow-up experiment. Stage 2 evaluated 0, 0.5, 1, and 2 mM using semen from five roosters and six separately prepared pooled-sample replicates per concentration. Samples were pre-cooled to 4°C, equilibrated with glycerol, and cryopreserved by horizontal exposure to liquid nitrogen vapor (4 cm above the liquid phase for 10 min, approximately 15-20°C/min) before immersion into liquid nitrogen (-196°C). Then, after thawing, the samples were evaluated for viability, motility parameters, and membrane integrity. In the second stage of the experiment, analysis of variance (ANOVA) revealed significant differences among groups for overall motility (p < 0.001), progressive motility (p < 0.001), membrane integrity (p = 0.026), and sperm viability (p = 0.044). Subsequent post-hoc analysis using Duncan’s multiple range test showed that the 0.5 mM crocin group exhibited significantly higher overall motility compared with the control, 1 mM, and 2 mM groups.In Stage 2, 0.5 mM crocin produced the highest total motility, progressive motility, and viability, whereas 1 mM produced the highest membrane integrity. These endpoint-specific findings identify 0.5 mM as a promising concentration for preserving the measured post-thaw sperm-quality parameters; confirmation using independent rooster-level replication and fertility outcomes is warranted.

Keywords

Sperm

Crocin

Freezing

Rooster

Subjects

• Reproductive

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