Preliminary in vitro effects of trehalose, pentoxifylline and MitoTEMPO on post-thaw quality of ram semen

Background Ram semen cryopreservation is associated with reduced post-thaw sperm quality, partly due to membrane destabilization and oxidative stress. This preliminary in vitro study compared trehalose, low-dose pentoxifylline, and MitoTEMPO, used individually or in combination, as additives for ram semen cryopreservation. Semen collected from six healthy Merino rams during the breeding season was pooled on each collection day and divided into eight treatment groups: control, trehalose (50 mM), pentoxifylline (0.25 or 0.5 µM), MitoTEMPO (0.025 or 0.05 mM), and two MitoTEMPO plus pentoxifylline combinations. This procedure produced eight pooled ejaculates per treatment group. After freezing and thawing, sperm motility, plasma membrane integrity, acrosome integrity, capacitation status, total antioxidant capacity, and total oxidant status were evaluated. Results Trehalose and pentoxifylline at 0.5 µM significantly improved several post-thaw sperm quality traits. Compared with the control group, trehalose significantly increased post-thaw motility from 31.25 ± 4.43% to 42.50 ± 5.35%, plasma membrane integrity from 53.78 ± 2.47% to 65.29 ± 4.14%, and acrosome integrity from 54.66 ± 4.41% to 69.69 ± 1.48% ( P < 0.05). Pentoxifylline at 0.5 µM also resulted in higher motility (40.63 ± 4.96%), plasma membrane integrity (64.24 ± 2.09%), and acrosome integrity (64.56 ± 2.69%) than the control group ( P < 0.05). The overall treatment effect was significant for total oxidant status ( P = 0.037) and total antioxidant capacity ( P < 0.001). However, Sidak-adjusted pairwise comparisons did not identify significant differences between individual treatment groups for total oxidant status. MitoTEMPO alone did not significantly improve the main post-thaw sperm quality traits compared with the control group, and the MitoTEMPO plus pentoxifylline combinations did not provide a significant additional benefit. Capacitation status did not differ significantly among groups ( P = 0.178). Conclusions Trehalose and pentoxifylline at 0.5 µM improved several in vitro post-thaw sperm quality traits and total antioxidant capacity of cryopreserved ram semen. Because the study used pooled ejaculates and did not include fertility trials, these findings should be interpreted as preliminary and require confirmation using individual ejaculates, objective sperm motion analysis, and fertility outcomes.