Pregnancy outcomes after TAI with cooled versus frozen bovine semen: A meta-analysis

Bovine cooled semen (CS) has been selectively re-incorporated as a mitigating strategy to frozen semen (FS) in timed artificial insemination (TAI) programs, raising questions about the biological limits of cryopreserved sperm cells in synchronization protocols. This meta-analysis evaluated the impact of semen type and selected management-related factors on P/AI in bovine TAI. Fourteen studies encompassing 8785 inseminations and 3816 pregnancies were analyzed following PRISMA guidelines. Risk difference (RD) was used to compare P/AI between CS and FS. Overall, CS showed a tendency toward greater P/AI (RD = 2.95%; 95% confidence interval [CI]: -2.75-8.64; P = 0.075), with substantial heterogeneity (I² = 81.8%). The most pronounced differences favoring CS were observed in cows without expression of estrus (RD = 17.1%; 95% CI: 8.9-25.4%) and when 0.25 mL straws were used (RD = 6.4%; 95% CI: 2.1-10.6%). In parallel, CS better preserved average path velocity (VAP), a sperm trait highly sensitive to cryoinjury. Exploratory meta-regression indicated that differences between semen types were partly driven by biological and procedural factors, with estrus expression as the main source of heterogeneity. Collectively, these findings indicate that fertility outcomes with FS in TAI programs are particularly vulnerable when ovulation timing deviates from optimal synchrony, reflecting a reduced overlap between sperm longevity and ovulation. While FS remains the standard for large-scale application, refinements in semen preservation, handling practices, and ovulation control may be required to improve fertility consistency under suboptimal physiological conditions.