Facultad de Ciencias Veterinarias, Universidad Nacional de Asunción, San Lorenzo, Paraguay; Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Naples, Italy.
Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Naples, Italy.
Theriogenology. 2024 Nov;229:118-126. doi: 10.1016/j.theriogenology.2024.08.024. Epub 2024 Aug 21.
Freezing-thawing procedures and semen manipulation for in vitro fertilization induce oxidative stress, which in turn leads to impaired sperm quality. The aim of this study was to evaluate whether incubation of frozen-thawed buffalo semen with olive fruit extracts (OFE), known to contain a high concentration of phenolic antioxidants, would improve semen quality by reducing oxidative stress. Frozen sperm (4 ejaculates/4 bulls/3 replicates) were thawed and diluted to 30 × 10/mL in IVF medium with 0, 72, 143, and 214 μL/mL of OFE, corresponding to 0 (D0-control), 50 (D50), 100 (D100), and 150 (D150) μM hydroxytyrosol. Sperm viability, acrosome integrity, membrane functionality, motility, and sperm kinetics were evaluated immediately after thawing (T0) and after 1 (T1) and 2 h (T2) of incubation at 38.7 °C. Based on the results, sperm biological antioxidant potential (BAP) and ROS levels (ROMs) were assessed in D0 and D100 groups at T1 and T2. To assess the effect of OFE on fertilizing ability, heterologous penetration rates were also evaluated, using bovine abattoir-derived oocytes. The treatment with OFE at all concentrations tested increased (P < 0.05) the percentage of acrosome intact spermatozoa compared to the D0-control at T1, but the effect was more evident (P < 0.01) with D100 (54.5 ± 3.0, 60.5 ± 1.5, 65.2 ± 3.3, and 62.5 ± 1.7, with D0, D50, D100, and D150 OFE, respectively). Total motility, progressive motility, rapid velocity, and progressive velocity decreased (P < 0.05) at T2 only in the D0-control group. The percentage of rapidly progressive sperm and the progressive motility tended to increase (P < 0.10) at T1 and T2, respectively, in D100 compared to D0 (24.7 ± 4.1 vs 16.4 ± 1.6 and 22.8 ± 2.7 vs 17.0 ± 1.2, respectively). The treatment with D100 OFE of frozen-thawed sperm increased (P < 0.05) some kinetic parameters (VAP and WOB). Spermatozoa incubated with D100 OFE exhibited higher (P < 0.01) total and normospermic oocyte penetration rates compared to D0 (86.5 ± 1.4 vs 78.5 ± 0.7, and 70.6 ± 1.5 vs 63.8 ± 1.1, respectively). Additionally, D100 OFE increased sperm BAP concentrations at both T1 and T2, while ROS levels were unaffected. These results suggest that incubating frozen-thawed buffalo semen with OFE is an effective strategy for preserving semen quality and in vitro fertilization ability by enhancing sperm antioxidant capacity.
冷冻-解冻程序和精液操作会导致体外受精产生氧化应激，进而导致精子质量受损。本研究旨在评估在冷冻-解冻水牛精液中添加橄榄油提取物（OFE）是否可以通过减少氧化应激来改善精液质量。将 4 个公牛的 4 个样本的冷冻精液（每个样本 3 个重复）解冻并稀释至 30×10/mL 的 IVF 培养基中，OFE 的浓度分别为 0（D0-对照）、50（D50）、100（D100）和 150（D150）μL/mL，相应的羟基酪醇浓度为 0、50、100 和 150μM。在解冻后立即（T0）以及孵育 1（T1）和 2（T2）小时后，评估精子活力、顶体完整性、膜功能、运动性和精子动力学。根据结果，在 T1 和 T2 时，在 D0 和 D100 组中评估了精子生物抗氧化潜力（BAP）和 ROS 水平（ROMs）。为了评估 OFE 对受精能力的影响，还使用牛屠宰场来源的卵母细胞评估了异种穿透率。与 D0 对照组相比，在 T1 时，所有测试浓度的 OFE 处理均增加了顶体完整精子的百分比（P<0.05），但 D100 的效果更为明显（P<0.01）（分别为 54.5±3.0、60.5±1.5、65.2±3.3 和 62.5±1.7，D0、D50、D100 和 D150 OFE）。只有在 D0 对照组中，T2 时总运动、渐进运动、快速速度和渐进速度才会降低（P<0.05）。与 D0 相比，在 T1 和 T2 时，D100 中的快速进展精子百分比和渐进运动趋势增加（P<0.10）（分别为 24.7±4.1 比 16.4±1.6 和 22.8±2.7 比 17.0±1.2）。用 D100 OFE 孵育冷冻-解冻的精子增加了一些运动参数（VAP 和 WOB）。与 D0 相比，用 D100 OFE 孵育的精子总穿透率和正常精子穿透率更高（P<0.01）（分别为 86.5±1.4 比 78.5±0.7 和 70.6±1.5 比 63.8±1.1）。此外，D100 OFE 在 T1 和 T2 时均增加了精子 BAP 浓度，而 ROS 水平不受影响。这些结果表明，用橄榄油提取物孵育冷冻-解冻的水牛精液是一种有效的策略，可以通过增强精子抗氧化能力来保持精液质量和体外受精能力。
