Supplementation of cryopreservation medium with pyrroloquinoline quinone improves post-thaw sperm quality in rams and fertility outcomes in ewes.

Cryopreservation allows long-term storage and global distribution of high-genetic-merit ram sperm, but the freezing-thawing process damages sperm cells and lowers their fertilizing ability. This work assessed how different levels of pyrroloquinoline quinone (PQQ) supplementation affect frozen-thawed ram sperm characteristics and fertility results. Semen samples received 0, 10, 100, 1000, or 10,000 nM PQQ in the extender before standard freezing. Post-thaw evaluations measured sperm movement, membrane integrity, morphology, mitochondrial status, acrosome condition, cell survival, apoptosis markers, DNA damage, ROS levels, lipid peroxidation, and reproductive outcomes. Among all groups, 100 nM PQQ was most effective (P ≤ 0.05), improving total motility (60.5%), progressive motility (40.2%), membrane functionality (61.5%), high mitochondrial membrane potential (65.7%), acrosome integrity (68.0%), and viability (51.5%), while reducing late apoptosis-like/membrane-damaged spermatozoa (38.0%), DNA fragmentation (9.5%), DCF-positive spermatozoa (20.1%), and lipid peroxidation (3.50 nmol/mL) compared with controls and other PQQ levels. Fertility testing also showed that 100 nM PQQ produced higher pregnancy (50%), parturition (48%), and lambing (50.5%) rates than the frozen-thawed untreated control (25%, 23.5%, and 24.5%, respectively). Thus, the optimal PQQ dose (100 nM) maintains post-thaw sperm quality and fertility, making it a valuable cryopreservation additive for sheep breeding.