The widespread use of artificial insemination (AI) has made it the leading method for efficiently transferring desirable bull genetic traits. Particularly in the dairy industry, it is widely recognized as an essential tool for improving herd genetics while reducing the transmission of venereal diseases [1]. Steady genetic progress in dairy cattle can largely be attributed to continuous advancements in semen preservation technologies, which have significantly improved the reliability and efficiency of AI. Research in this area flourished in many countries, first with cooled liquid-preserved semen (LS) and later with frozen semen (FS) [2]. Currently, the use of FS for AI in cattle reproduction is considerably more prevalent, accounting for 95 % of cases, whereas LS is utilized less frequently [3,4].

However, LS preservation provides notable advantages in comparison with cryopreservation, primarily by avoiding the stressors associated with freezing and thawing, including damage induced by extreme low temperatures and osmotic imbalances affecting spermatozoa [5]. During cryopreservation, about 40–50 % of the sperm population does not survive the freezing procedure, which results in considerably higher preservation losses [6]. For instance, fresh AI doses may require only one-tenth the number of sperm needed for a frozen semen dose, depending on sperm quality [7]. From the perspective of sustainable utilization of animal resources, economic efficiency of AI centers, lower storage costs, and optimized use of high demand breeding bulls, LS may present a possible alternative to FS. Moreover, Wiebke et al. demonstrated that insemination with LS resulted in a higher pregnancy per AI (P/AI) compared to insemination with FS, with a success rate of 45.4 % for LS vs. 33.7 % for FS [8]. This difference may be attributed to the potentially longer lifespan of LS in the female reproductive tract, as suggested by Bucher et al. [9].

The adoption and utilization of LS in German dairy farms, as well as globally, remain limited, likely due to the lack of evidence-based recommendations for its handling [10]. In practice, a broad spectrum of cooling protocols and holding temperatures is used, often leading to inconsistent results in sperm quality and viability. Reported storage temperatures vary widely, ranging from 5 °C, 10 °C–15 °C, and even room temperature [4]. Unlike semen processing in boars and stallions, no studies have been published on cooling rates for LS in bulls [4]. Consequently, the optimal preservation approach for LS remains inadequately defined and requires further investigation. Previous research in other species has demonstrated the positive effect of a gradual cooling to 5 °C in liquid-preserved boar semen [11]. Based on these findings, we hypothesized that a gradual cooling protocol incorporating a 6 h holding phase at 16 °C could exert a beneficial effect on sperm quality. The application of a gradual cooling curve was used to evaluate this hypothesis (Protocol C, as described in Materials and Methods).

Currently, the egg yolk-based extender Caprogen is the most widely used medium and remains the gold standard for LS preservation [4]. It is generally stored at a temperature of 16 °C [12]. However, growing biosecurity concerns and the imperative of disease control in international semen trade have raised suspicions that egg yolk-based extenders may act as vectors for the transmission of various pathogens, including Escherichia coli, Staphylococcus spp., Streptococcus spp., Pseudomonas spp., Haemophilus spp., Salmonella spp., Avian influenza virus, Campylobacter spp., Listeria spp., and Mycoplasma spp. [13]. Additionally, the presence of foreign particles in egg yolk complicates semen evaluation and quality control [14]. The aim of eliminating animal-derived components from extenders and reducing the residual risk of contamination associated with egg yolk prompted the evaluation of a fully synthetic extender (FSE) in this study.

Vishwanath and Shannon highlighted the limited shelf life of 2.5–3 days post-collection, emphasizing the challenges of short-term storage as fundamental reasons for the restricted use of LS [1]. The consideration of extending the storage duration of LS is partly based on the observation that bovine spermatozoa can be stored in the epididymis for several weeks without negatively affecting their fertilization capacity [15]. This study combines three key perspectives - standardized handling (a), evaluation of a fully synthetic extender (b), and extended storage duration (c) - with the goal of identifying optimal conditions, including extender composition, cooling protocol, and storage temperature, to prolong the preservation period of LS and enhance its practical applicability for widespread use.