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Fewer studies investigate the effects of underlying genetic factors related to semen characteristics, significantly affecting sheep farm profitability. This study aimed to identify single nucleotide polymorphisms (SNP) and genomic regions associated with fresh and frozen-thawed semen traits in rams with low (Hasak) and high (Hasmer) cryotolerance. Semen collected from 11 (5 Hasak with low and 6 Hasmer with high cryotolerance) rams cryopreserved in 0.25 ml straws in the breeding season. Quality characteristics were determined in fresh, equilibrated, and frozen-thawed semen. A Genome-Wide Association Study (GWAS) was conducted to unveil the genetic structure that might be attributed to cryotolerance in low and high cryotoleranced rams. Fresh (regarding total and progressive motility) and equilibrated semen quality were similar in Hasak and Hasmer rams (p > 0.6). However, the freeze-thawing process had a more pronounced negative effect on ram semen traits in Hasak than in Hasmer (p < 0.05). GWAS revealed 27 SNPs correlated with post-thaw semen parameters. Moreover, network analyses revealed pathways related to sperm ion channels and their activities, providing insights into the intricate molecular mechanisms underlying sperm physiology and emphasizing their role in potentially impacting sperm cryotolerance. The functional significance of detected SNPs and the associated pathways require further exploration., Competing Interests: Conflict of interest statement None of the authors have any conflict of interest to declare., (Copyright © 2025 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

To improve the fertility of cervical artificial insemination (AI) in sheep, we investigated isoxsuprine HCl usage on the cervical passage during cervical AI. We also compared cervical and laparoscopic AI fertility results of using chilled semen at different durations. Semen was collected from rams and diluted as 20 × 10 6 or 400 × 10 6 spermatozoa/straw for laparoscopic and cervical AI, respectively, and chilled to 4°C within 2 h. Sheep were inseminated with chilled semen for 8 or 24 h via the laparoscopic or cervical AI method. Moreover, some of the cervical inseminated sheep were injected intramuscularly with 0.5 mg/kg of isoxsuprine HCl 15 min before AI. As a result, the use of isoxsuprine HCl did not affect cervical transit and fertility. In addition, fertility was affected by the storage duration of the semen; laparoscopic AI was more successful than cervical AI in terms of fertility; if cervical AI is performed, the duration between semen collection and AI should be less than 8 h after chilling the semen at 4°C, and if laparoscopic AI is performed, the time between semen collection and insemination can be up to 24 h after chilling the semen at 4°C. Longer storage periods should be studied., (© 2024 The Author(s). Animal Science Journal published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Animal Science.)