Document Type : Research Paper

• Animal Physiology

Extended Abstract

Introduction

Trehalose, a non-reducing disaccharide with membrane-stabilizing and antioxidant properties, has shown protective effects in sperm preservation across various species. Its mechanisms of action are primarily due to the prevention of ice crystal formation, reduction of oxidative stress, and stabilization of the plasma membrane. However, the optimal concentration of trehalose appears to vary between species and extender types. Despite extensive research on mammalian sperm cryopreservation, there is limited information on the effects of trehalose on poultry semen, particularly rooster sperm. The aim of the present study was to evaluate the effect of two concentrations of trehalose (50 and 100 mM) added to Ringer’s extender on sperm quality parameters of aging Ross roosters stored for 24 hours at 5 °C.

Materials and Methods

The study was carried out on ten mature Ross roosters (45 weeks of age). Birds were kept under controlled environmental conditions for two weeks prior to semen collection, with ambient temperature maintained at 19–23 °C and a photoperiod of 14 hours light and 10 hours dark. Feed and water were provided ad libitum in accordance with Ross management guidelines. Semen was collected by abdominal massage every three days in four separate sessions. Immediately after collection, ejaculates were placed in pre-warmed tubes (38 °C) and transferred to the laboratory. After initial evaluation, ejaculates with poor quality were discarded, and only samples meeting the required standards were pooled. Each pooled sample was diluted at a ratio of 1:20 with Ringer’s solution and divided into three experimental groups: control (without trehalose), extender supplemented with 50 mM trehalose, and extender supplemented with 100 mM trehalose. Diluted semen samples were stored at 5 °C for 24 hours, and assessments were performed at 0, 6, 12, and 24 hours of storage. The parameters measured included total motility and progressive motility (evaluated under phase-contrast microscopy, 40× magnification), viability (eosin-nigrosin staining), plasma membrane integrity (hypo-osmotic swelling test), and morphological abnormalities. The experiment was conducted in a completely randomized design with three treatments and four replicates. Data were analyzed using the GLM procedure of SAS software. Least square means were compared using Duncan’s multiple range test, and differences were considered significant at P < 0.05.

Results and Discussion

The addition of trehalose significantly influenced sperm quality parameters during storage. The 50 mM trehalose treatment consistently preserved higher values of total and progressive motility compared with both the control and the 100 mM group (P < 0.05). This effect was particularly evident at 12 and 24 hours of storage, where the differences between treatments were most pronounced. In contrast, the 100 mM trehalose treatment in some cases presented a negative trend on sperm morphology. Sperm viability was markedly enhanced in the 50 mM group, with higher percentages of live spermatozoa throughout the storage period compared to the other treatments. Similarly, plasma membrane integrity was best maintained with 50 mM trehalose, confirming its protective role in preserving sperm structural functionality. The incidence of morphological abnormalities was also significantly reduced in the 50 mM group. The findings of this study clearly demonstrate the protective role of trehalose at the optimal concentration of 50 mM in preserving rooster sperm during short-term storage at 5 °C. The beneficial effects of trehalose can be attributed to its ability to stabilize the lipid bilayer of the sperm plasma membrane, prevent osmotic stress, and reduce lipid peroxidation. These results are consistent with previous reports in mammalian species such as bull, ram, and rabbit, where trehalose supplementation improved post-storage sperm quality. However, the lack of beneficial effects at 100 mM trehalose suggests that excessive concentrations may induce hyperosmotic stress, leading to detrimental impacts on spermatozoa. Such concentration-dependent responses highlight the importance of optimizing trehalose levels for each species and storage condition. This study also underlines the potential of trehalose as a non-toxic, cost-effective additive that could be incorporated into rooster semen extenders to enhance fertility outcomes in artificial insemination programs. Nevertheless, further research is necessary to investigate fertility performance following insemination with trehalose-treated semen and to compare trehalose with other protective agents under different extender formulations and storage conditions.

Conclusion

In conclusion, supplementation of Ringer’s extender with 50 mM trehalose significantly improved semen quality in aging Ross roosters during 24-hour storage at 5 °C. This concentration effectively preserved motility, viability, plasma membrane integrity, and reduced morphological abnormalities compared with the control. The higher concentration (100 mM) was not beneficial and may even have adverse effects. These results suggest that 50 mM trehalose can be recommended as an effective additive for short-term storage of rooster semen. Future studies should focus on fertility trials, longer storage periods, and interaction effects with other extender components to validate the practical applications of these findings.

Author Contributions

Saleh Tabatabaei Vakili: Conceptualization, Methodology, Supervision, Writing-Review & Editing

Safoura Barzegar:  Investigation, Software, Formal analysis, Witing-Original draft

Data Availability Statement

Data available on request from the authors

Acknowledgements

The authors gratefully acknowledged the Agricultural Sciences and Natural Resources University of Khuzestan for supporting and funding this research.

Ethical considerations

The study was approved by the Ethics Committee of Agricultural Sciences and Natural Resources University of Khuzestan. The authors avoided data fabrication, falsification, plagiarism, and misconduct.

Conflict of interest

The author declares no conflict of interest.