The impact of varying doses of moringa leaf methanolic extract supplementation in the cryopreservation media on sperm quality, oxidants, and antioxidant capacity of frozen-thawed ram sperm

Abstract

To increase rams' post-thaw semen quality following cryopreservation, this study used enriched Tris-based diluent with varying amounts of moringa leaf methanolic extract (MLME). The antioxidant activity, total phenolic, and total flavonoid content were all assessed in MLME. The sperm of five healthy Awassi rams were collected, divided into 4 equal aliquots, and diluted [1:5; (v/v)] in Tris-citrate-glucose extender supplemented with 0.48, 0.56, and 0.64 mg MLME/ml or without MLME supplementation (control). The percentages of sperm total motility (STM, %), sperm progressive motility (SPM, %) and viability (V, %), abnormal morphology (AM, %), membrane functional integrity (MFI, %), and acrosome integrity (AI %) were measured. Malondialdehyde (MDA), nitric oxide (NO), ascorbic acid (AA), superoxide dismutase (SOD), glutathione peroxidase (GPx), total cholesterol (TC), low-density lipoproteins (LDL), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), zinc (Zn), and copper (Cu) were measured. The total phenolic gallic acid and flavonoid catechin (equivalent) contents were 19.78 mg/g and 11.94 mg/g, respectively. 2,2-Diphenyl-1-picrylhydrazyl (34.37 mM TE/g) and 2,2'-azino-bis/3-ethylbenzothiazoline-6-sulfonic acid (53.47 mM TE/g) were found in MLME. MLME had a 64.59 mM TE/g ferric-reducing power. In comparison to control, the addition of 0.64 mg/ml MLME to Tris-based extender resulted in the highest (P < 0.001) STM (55.22 ± 0.98), SPM (45.41 ± .70), SV (60.01 ± 1.05), MFI (75.23 ± 0.77), and AI (73.13 ± 0.72) and the lowest (P < 0.001) AM (21.34 ± 0.72) values. In comparison to the control, the addition of 0.56 mg/ml semen extender resulted in lower STM, SPM, SV, MFI, and AI with higher AM percentages. MDA (P = 0.03), NO (P = 0.012), CHO (P = 0.0001), and LDL (P = 0.004) were reduced by 0.64 mg/ml MLME, while AA (P = 0.017) and SOD (P = 0.0001) were elevated. In conclusion, the highest copper (P = 0.006) and lowest zinc concentrations in MLME (0.48 mg/ml extender) deteriorated the post-thaw semen quality, prompting us to suggest the addition of 0.64 mg MLME to rams' Tris-based semen extender.

Keywords: MLME; Oxidants–antioxidants; Post-thaw semen quality; Rams; Trace minerals.