Using an efficient method to separate sperms with low and high motility in fresh stallion semen treated with Resiquimod (R848), aiming to separate X and Y chromosome-bearing sperms

Document Type : Original Article

Authors
Seyed Pourya Hosseyni 1
Ali Kadivar 2
Naser Shams Esfandabadi 3
Najmeh Davoodian 4
Seyed Abbas Mirzaei 5
1 DVSc Student of Theriogenology, Department of Clinical Sciences, Faculty of Veterinary Medicine Shahrekord University, Shahrekord- Iran.
2 Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord- Iran
3 Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord-Iran
4 Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord- Iran
5 ellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord- Iran
10.22034/ijvcs.2025.15266.1143
Abstract
The aim of the present study was to develop and optimize an efficient method to separate sperms with low and high motility in fresh stallion sperm treated with resiquimod (R848), aiming to separate X and Y chromosome-bearing sperms. Fresh semen samples were collected from three different stallions in triplicate. For 30 million sperm, swim-up was conducted in HTF medium for 30 minutes. Finally, five distinct layers were separated from the top to the bottom of the medium. Four doses of 0.06 (D0.06), 0.12 (D0.12), 0.16 (D0.16) and 0.24 (D0.24) µl of resiquimod at a concentration of 3 µM were tested and compared with control groups without resiquimod in terms of sperm motility parameters. Doses D0.12, D0.16, and D0.24 induced a significant reduction in sperm velocity-related indices across most harvested layers (P<0.05). For dose D0.16, VSL in the second and third layers (from top to bottom) of the treatment group showed a significant increase compared to the control group (P<0.05). Additionally, dose D0.16 caused a significant decrease in sperm motility indices in the two lower layers (P<0.05). At the highest harvested layer of this dose, the VSL in the treatment group was significantly higher than in the control group (P<0.05). However, the indices VCL and VAP showed no significant differences between the treatment and control groups (P>0.05). Overall, the best dose used in this study that produced the most reasonable changes in sperm motility indices in the harvested layers was D0.16.

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Iranian Journal of Veterinary Clinical Sciences
Volume 19, Issue 1 - Serial Number 32
Spring & Summer 2025
August 2025
Pages 3-13