Semen diluents are crucial for the effective utilization of male germplasm resources and for enhancing the efficiency of artificial insemination (AI). However, reports on the in vitro low-temperature preservation of semen are still limited. This study comprised three separate experiments. Experiment 1 (semen storage): An ex vivo experiment using one ejaculate from each of five bulls, split into two aliquots per ejaculate, stored at 0 °C for 15 days, with repeated assessments of sperm quality (n = 5 biological replicates). Experiment 2 (IVF): An in vitro fertilization study using slaughterhouse-derived oocytes fertilized with day-15 stored semen from Experiment 1, conducted in five independent sessions (n = 5 sessions). Experiment 3 (AI): A non-randomized interventional field study involving 400 synchronized cows at China Hualing Ranch, allocated upon detection of estrus to receive either treatment or control semen (n = 400 cows). The primary outcome for Experiment 1 was total motility at day 15; for Experiment 2, blastocyst formation rate per oocyte; for Experiment 3, pregnancy rate at day 50 post-AI among all allocated cows. These findings suggest that the treatment formulation may offer advantages for liquid storage of bull semen at 0 °C.