Enhancing Rahmani Ram Semen Cryosurvival Through Oral Moringa Oil and Microencapsulation: In Vivo and In Silico Mechanistic Insights.

Abstract

Cryopreservation of ram semen is challenged by the high polyunsaturated fatty acid content in spermatozoa, which leads to increased oxidative stress and cellular damage. This study explored the potential of orally administered moringa oil (MO) or its microencapsulated form (MON) to protect ram spermatozoa during cryopreservation by assessing their effects on semen quality, antioxidant capacity, apoptosis, seminal metabolic enzyme activity, as well as molecular docking study. Fifteen Rahmani rams were randomly split into three groups (n = 5 per group) and fed a basal diet. The control group (CON) received 1 mL of distilled water orally, while the second and third groups received 2 mL of MO or 1 mL of MON, respectively, daily for 4 months. Semen samples were collected bi-weekly using an artificial vagina, pooled, extended and cryopreserved following standard protocols. Results demonstrated significantly higher (p < 0.05) post-thaw sperm viability, progressive motility and membrane integrity in the MO group compared to other groups after equilibration (at 5°C for 4 h), post-thawed ram semen (at 37°C for 30 s) or incubated at 37°C and 5% CO₂ for 2 h. Regarding apoptotic sperm, the orally administered MO group had a significantly greater number of viable spermatozoa (p < 0.001) than the other groups. Although all treated groups had a lower percentage of early apoptosis than the control, MON administration resulted in a significant increase in the percentages of necrotic sperm compared to the MO group (p < 0.05). The TAC was highest and MDA was lowest (p < 0.05) in the MO group. Molecular docking analysis revealed the binding energies (kcal/mol) of bioactive compounds from MO including apigenin, ferulic acid and naringenin with three target proteins: ADAM17 (-4.47, -4.49 and -4.88, respectively), DNase1 (-4.42, -3.47 and -4.46, respectively) and SHBG (-6.38, -4.70 and -6.43, respectively). These findings indicate that orally administering MO has a more pronounced positive effect on Rahmani ram semen quality, apoptosis, and antioxidant status following cryopreservation compared to its microencapsulated form.