Direct cervical thawing of frozen semen simplifies artificial insemination without compromising fertility in dairy cattle

Artificial insemination (AI) remains a fundamental tool for improving reproductive efficiency and accelerating genetic progress in dairy cattle production systems. Despite major advances in semen cryopreservation and reproductive management, the basic procedural steps involved in AI have remained largely unchanged for decades. Conventional protocols require semen-thawing in a water bath prior to insemination (Traditional thawing, TT), introducing additional handling steps that may expose spermatozoa to temperature fluctuations and increase procedural complexity during field operations. This study evaluated a simplified AI approach in which frozen semen straws were inseminated directly from liquid nitrogen storage, omitting the conventional thawing step (Cervical thawing, CT). Semen from a single bull was used in all experiments, with straws randomly allocated to each treatment (n = 25, 10, and 96 straws per treatment for sperm quality, mitochondrial activity, and field trial components, respectively). Sperm functional parameters were evaluated in vitro, including motility, membrane integrity, acrosome integrity, viability, and mitochondrial activity using fluorescence microscopy and flow cytometry. In addition, a two-year field trial was conducted to obtain preliminary fertility data under dairy farm conditions. No significant differences were observed between treatments for most sperm quality parameters, although progressive motility was slightly greater in the TT (TT: 19.5% ± 2.9 vs. CT: 16.5% ± 2.3; P = 0.02). Mitochondrial activity increased under CT (TT active: 23.00% ± 6.53 vs. CT active: 50.1% ± 7.59; P = 0.0043), and conception rates were comparable between treatments in the field trial (P = 0.386). These findings suggest that direct cervical thawing preserves key sperm functional characteristics while eliminating a procedural step in the insemination process. Although the fertility data are preliminary, the agreement between laboratory and field observations suggests this simplified protocol may represent a practical alternative to conventional semen handling procedures. By reducing handling time and procedural complexity, this approach could offer operational advantages for AI programs in dairy production systems. Further studies in larger commercial herds will be necessary to confirm the reproductive performance and operational benefits of this protocol under routine breeding conditions.