Efficient transcervical artificial insemination (AI) in sheep remains a significant reproductive challenge, primarily due to the complex and highly convoluted structure of the ovine cervix. The cervical canal is narrow and tortuous, with multiple caudally oriented folds and rings that vary among animals. These anatomical features markedly hinder catheter passage, particularly in nulliparous or early post-partum ewes and in certain breeds [[1], [2], [3]]. Consequently, even when estrus synchronization protocols are widely applied, fixed-time AI often fails to achieve adequate cervical dilation at the time of insemination, thereby limiting fertility outcomes under field conditions [4,5].

Cervical softening is driven by extensive remodeling of the extracellular matrix (ECM), a process largely regulated by matrix metalloproteinases (MMPs)—particularly MMP-2—which degrade collagen and other structural components, thereby promoting tissue relaxation [[6], [7], [8], [9]]. In sheep, previous work has shown that carbetocin (Cb), a long-acting oxytocin receptor agonist, increases latent (L) MMP-2 activity in cervical explants and enhances cervical penetrability when administered systemically near the expected time of AI [10,11]. Additionally, intravaginal prostaglandin E2 (PGE2) has been demonstrated to facilitate cervical softening through MMP activation and ECM remodeling in ruminants [12,13], consistent with the PGE2-related effects reported in ovine cervical tissue [14].

Dimethyl sulfoxide (DMSO) is an amphipathic solvent widely used in biomedical and pharmacological research because of its ability to enhance membrane permeability and facilitate intracellular drug delivery [15,16]. Beyond its role as a vehicle, DMSO can modulate cellular signaling pathways and matrix metalloproteinase (MMP) activity, thereby influencing ECM remodeling in several tissues [17,18]. In ovine cervical explants, García-Barcelo et al. [14].reported that DMSO increased MMP-2 activity, suggesting a potential direct influence on cervical collagen turnover. Although these in vitro findings indicate a modulatory role for DMSO in cervical physiology, its in vivo effects on ECM dynamics and cervical penetrability in sheep remain poorly characterized.

The present study aimed to investigate the individual and combined effects of DMSO and Cb on cervical softening mechanisms in sheep, using both in vitro and in vivo approaches. First, we evaluated the effect of increasing concentrations of DMSO, alone or in combination with Cb, on MMP-2 and MMP-9 activity in cultured cervical explants from ewes in the follicular phase. Second, we assessed the in vivo impact of intravaginal DMSO, administered alone or co-administered with intramuscular Cb, on cervical penetrability during a standard ovulation synchronization protocol.

We hypothesized that DMSO would modulate cervical MMP activity and, when combined with Cb, might synergistically enhance cervical relaxation at the optimal time for AI. These findings may provide valuable insights for improving transcervical AI protocols in sheep.