冷冻保存是保护不同物种遗传物质的关键技术,尽管其固有的挑战与诱导精子损伤有关,细胞凋亡和脂质过氧化(LPO)。鉴于精子对LPO的抗氧化防御不足,人们对将额外的添加剂整合到补充剂中以改善哺乳动物精液质量越来越感兴趣。在这些添加剂中,黄酮类化合物由于其有效的抗氧化特性而引起了相当大的关注。因此,我们的研究旨在评估在冷冻保存培养基中补充黄酮(FL)和3-羟基黄酮(3-OH=)的功效,以保护犬精子免受冷冻的破坏性影响,并确保其生殖潜力的保持.从五只比格犬收集精液,然后合并。然后,样本分为7组,每个用1)0mM处理,2)0.1mMFL,3)0.2mMFL,4)0.4mMFL,5)0.1mM3-OH=,6)0.2mM3-OH=,7)0.4mM3-OH=。将精液样品在法国吸管中冷冻保存,并将甘油作为冷冻保护剂。在冷冻解冻的精液中,通过CASA系统和精子膜完整性的精子运动参数,顶体状态,线粒体活性,DNA片段化,早期凋亡与获能,在解冻后(0小时)和解冻后4小时使用流式细胞术评估LPO。结果表明,解冻后0h,FL0.1和3-OH=0.2组中具有未受损顶体的活精子比例显着增加。在这个时间点,与FL0.1和0.2组相比,3-OH=0.1显著降低了DNA片段化指数(DFI)。然而,在接下来的4小时之后,与FL0.2和3-OH=0.1相比,3-OH=0.4表现出最低的(P<0.05)DFI。此外,3-OH=0.4显示与解冻后0.10h的FL相比,非凋亡和非获能精子的比例最高(P<0.05)。同时,同一组显示出凋亡和获能精子细胞的显着减少,在解冻后0小时和4小时。此外,3-OH=0.1(0h和4h)和0.2mM(4h)可显着提高解冻后无LPO的活精子的比例。在FL团体中,只有0.4FL显着增加了无LPO的活精子的百分比。未观察到测试物质对精子运动的显着影响,细胞膜完整性,或线粒体活动。这些发现强调了黄酮和3-羟基黄酮在冷冻保存过程中增强精子弹性的有希望的作用。表明它们对顶体损伤的保护功能,获能,细胞凋亡和脂质过氧化。
Cryopreservation is a pivotal technique in safeguarding genetic material across diverse species, despite its inherent challenges linked to induced spermatozoa damage, notably apoptosis and lipid peroxidation (LPO). Given the insufficient antioxidant defense of spermatozoa against LPO, there is a rising interest in integrating additional additives into extenders to ameliorate mammalian semen quality. Among these additives, flavonoids have garnered considerable attention due to their potent antioxidative properties. Hence, our study aimed to assess the efficacy of flavone (FL) and 3-hydroxyflavone (3-OH = ) supplementation in the cryopreservation medium to protect canine sperm against the damaging impacts of freezing and ensure the preservation of their reproductive potential. Semen was collected from five Beagle stud dogs and then pooled. Then, the sample was divided into 7 groups, each treated with 1) 0 mM, 2) 0.1 mM FL, 3) 0.2 mM FL, 4) 0.4 mM FL, 5) 0.1 mM 3-OH = , 6) 0.2 mM 3-OH = , 7) 0.4 mM 3-OH = . Semen samples were subjected to cryopreservation in French straws and glycerol as a cryoprotectant. In the frozen thawed semen, sperm motility parameters by CASA system and sperm membrane integrity, acrosome status, mitochondrial activity, DNA fragmentation, early apoptosis with capacitation, and LPO were assessed using flow cytometry just after thawing (0 h) and 4 h post thaw. Results reveal significant increase in the proportion of live spermatozoa with undamaged acrosomes in the FL 0.1 and 3-OH = 0.2 groups at 0 h post thaw. At this time point, 3-OH = 0.1 significantly reduced the DNA fragmentation index (DFI) compared to the FL 0.1 and 0.2 groups. However, after the next 4 h, 3-OH = 0.4 exhibited the lowest (P < 0.05) DFI compared to FL 0.2 and 3-OH = 0.1. Additionally, 3-OH = 0.4 showed the highest (P < 0.05) proportion of non apoptotic and non capacitated spermatozoa compared to FL 0.1 0 h post-thaw. Simultaneously, the same group demonstrated significant reduction in apoptotic and capacitated sperm cells, at 0 h and 4 h post-thaw. Moreover, 3-OH = at 0.1 (0 h and 4 h) and 0.2 mM (4 h) significantly enhances the proportion of live sperm without LPO post thaw. Whitin the FL groups, only 0.4 FL significantly increased the percentage of live sperm without LPO. No significant effect of the tested substances was observed on sperm motility, cell membrane integrity, or mitochondrial activity. These findings highlight the promising role of flavone and 3-hydroxyflavone in enhancing sperm resilience during cryopreservation, suggesting their protective function against acrosome damages, capacitation, apoptosis and lipid peroxidation.