Towards cervical AI of sheep with stored semen: latest insights and future developments

Artificial insemination (AI) is widely used in sheep breeding; however, fertility obtained with frozen-thawed ram semen following cervical (vaginal) insemination remains markedly low compared with fresh semen. This limitation contrasts with the high fertility achieved after laparoscopic intrauterine insemination, highlighting the central role of the ewe cervix as a selective barrier for frozen spermatozoa. This review examines current knowledge on the mechanisms underlying reduced fertility after cervical AI with frozen semen and proposes research avenues to improve reproductive outcomes. Despite cryotolerance of ovine sperm comparable to other ruminants, cryopreservation induces molecular and functional alterations that impair interactions with the female reproductive tract. While advances in semen freezing-such as antioxidant supplementation, lipid incorporation, and manipulation of seminal plasma-enhance post-thaw survival, they do not fully restore fertility, emphasizing the importance of female-side factors. Cervical morphology, uterine contractions, immune responses, and particularly cervical mucus properties influence sperm transport. Variations in mucin composition, glycosylation patterns, metabolite profiles, and microbiota composition are associated with breed-dependent differences in sperm survival and passage through the cervix. Emerging evidence suggests that targeted modulation of cervical mucus-potentially through dietary, oral, or local interventions-may represent a novel strategy to enhance fertility. Recent advances in imaging, 3D reconstruction, microfluidics, and organ-on-chip technologies provide unprecedented opportunities to model sperm-cervix interactions under physiologically relevant conditions. Integrating these tools with optimized cryopreservation strategies and a deeper understanding of cervical physiology is essential to overcome current barriers and improve the efficiency of cervical AI with frozen semen in sheep.