Trequinsin mitigates heat stress–induced impairment of bovine sperm function and embryo development

Global climate warming is increasingly associated with reduced reproductive efficiency in cattle, as elevated temperatures impair sperm function and subsequent embryo development. During summer breeding, artificially inseminated spermatozoa may be exposed to hyperthermic conditions within the female reproductive tract, highlighting the need for strategies that mitigate heat stress–induced functional decline. Because cyclic nucleotide signaling and CatSper-mediated Ca²⁺ influx—key regulators of sperm motility—are susceptible to thermal disruption, we evaluated trequinsin, a phosphodiesterase-3 inhibitor, as a potential protective agent. Dose–response testing using swim-up–selected bovine sperm demonstrated that trequinsin did not alter total or progressive motility under normothermic conditions (38.5 °C), whereas under acute heat stress (41 °C) it improved motility across tested concentrations, with maximal recovery observed at 20 µM and no additional benefit at 50 µM. Accordingly, 20 µM was used for subsequent analyses. A computer-assisted sperm analysis revealed that heat stress markedly reduced curvilinear velocity and straight-line velocity over 2 h, while trequinsin significantly attenuated these declines and maintained higher movement efficiency. Functional relevance was confirmed by in vitro fertilization, where trequinsin-treated heat-stressed sperm yielded higher cleavage and blastocyst rates than heat-stressed controls, although not fully reaching normothermic levels. Gene expression analysis showed reduced stress and pro-apoptotic signatures in resulting blastocysts, with profiles approaching those of non-heat-stressed controls. Collectively, trequinsin partially preserves sperm fertilizing capacity and embryo developmental competence under thermal stress, suggesting its potential as a pharmacological strategy to mitigate summer infertility in cattle.