الفهرس 
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Abstract
The current study was performed in the Physiology and Biotechnology Lab, Animal Production Department, Faculty of Agriculture, Mansoura University during the period from September 2021 to September 2023. The present study aimed to: (1) Evaluate the effect of adding two concentrations of ethanolic Portulaca oleracea leaf extract (POLE), as a natural antioxidant, in a native form or Nano formulation (POLENF) on increasing the antioxidant status of sperm medium to improve the sperm freezing ability of goat semen (1st experiment). (2) Explore the potential of chia methanolic extract (MCSE) in Tris-soybean lecithin extender of ram semen on physical characteristics, kinetic parameters, acrosome status, and apoptosis of sperm cells as well as antioxidant capacity, lipid peroxidation in sperm medium after thawing, and expression of antioxidant enzymes and caspase 3 in ram spermatozoa (2nd experiment). POL was extracted to POLE in 100% ethanol, while POLENF was prepared by hydrothermal squeezing. Chia seeds were extracted to MCSE in 95% methanol. In the 1st experiment of this study, five sexually mature Damascus bucks with a body weight of 60 to 70 kg and age of 2 to 4 years with acceptable semen quality were used for semen collection. In the 2nd experiment, sexually mature Rahmani rams (n=5, age of 12–14 mo, and weight of 55.0–60 kg) were used as semen donors. Semen was collected by artificial vagina (30 ejaculates in 1st EXP. And 35 ejaculates in 2nd EXP. The basic semen extender used in both experiments was Tris-soybean lecithin extender (TSBLE). In the 1st experiment, semen was diluted with five extender types as follows: E1 = control extender, E2 = control extender supplemented with POLE at 50 µg/mL extender, E3 = control extender supplemented with POLE at 100 µg/mL extender, E4 = control extender supplemented with POLENF at 50 µg/mL extender, and E5 = control extender supplemented with POLENF at 100 µg/mL extender. In the 2nd experiment, semen was diluted with five extender types as follows: E1 = control extender, E2 = control extender supplemented with MCSE at 125 µg/mL extender, E3 = control extender supplemented with MCSE at 250 µg/mL extender, E4 = control extender supplemented with MCSE at 375 µg/mL extender, and E5 = control extender supplemented with MCSE at 500 µg/mL extender. In both experiments, semen was evaluated after equilibration (5oC for 2 h) and thawing, and thawed semen incubated for 2 h at 37oC. The obtained results could be summarized as the following: First experiment: 1. Total flavonoids were higher in POLE than in POLENF. 2. Both extracts showed strong free radical scavenging capacity by DPPH assay, being higher in POLENF than in POLE, while antioxidant activity by FRAP assay had no differences. 3. After equilibration, thawing, and incubation, semen extended by POLENF (100 µg) significantly improved progressive motility, vitality, membrane integrity of spermatozoa in comparison with the free-extender. 4. In post-semen, the total antioxidant capacity was the highest, while MDA and H2O2 were the lowest in semen with POLENF (100 µg).5. The differences in AST, ALT, and LDH activity between the supplemented and free extenders were not significant. 6. Viable sperm percentage significantly increased, while early apoptotic sperm percentage significantly decreased by all supplements as compared to control. 7. Apoptotic sperm percentage significantly decreased by POLE at a high level (100 µg) and both levels of POLENF in comparing with control. 8. No significant differences were found in necrotic sperm percent between treatments. Nano-formulation of ethanolic extract of Portulaca oleracea leaves at a level of 100 µg/mL gave valuable results as an effective scavenger for free radicals during cryopreservation of goat semen. Second experiment: 1. Results revealed higher content of linolenic acid (67.5%), linoleic acid (17.99%), total phenolic and flavonoid compounds, and had higher antioxidant activity by DPPH and FRAP assays. 2. MCSE (375 and 500 µg/mL) increased sperm motility, livability, and membrane integrity after equilibration, thawing, and incubation. 3. MCSE (375 and 500 µg/mL) increased CASA analysis (DCL, DSL, DAP, VCL, VSL, VAP, ALH, BCF, STR, LIN, and WOB). 4. MCSE (375 and 500 µg/mL) increased percentage of live sperm with intact acrosome after thawing. 5. MCSE (500 µg/mL) increased viable sperm and decreased apoptotic spermatozoa after thawing. MCSE (250, 375, and 500 µg/mL) decreased caspas-3 level in semen after thawing. 6. MCSE (500 µg/mL) showed the highest TAC and the lowest MDA post-thawed sperm medium. 7. All MCSE levels, except at 125 µg/mL, increased gene expression of SOD1, CAT, GPx1, and GABPB1, while all MCSE levels decreased gene expression of CASP3. Supplementation of Tris-soya bean lecithin extender with methanolic extract of chia seeds, as a natural antioxidant at a level of 500 µg/mL, has vital roles in maintaining freezing ability of ram spermatozoa after cryopreservation. GENERAL CONCLUSION: In semen of small ruminants, extracts of natural antioxidants such as POLE (100 µg/mL) or POLENF (50 µg/mL) in extender of goat semen as well as MCSE (500 µg/Ml) in extender of ram semen are essential exogenous source to improve freezing ability which may has positive impact on sperm fertility in sheep and goats.