染料木素对牛精液冷冻保存效果的影响

doi:10.11843/j.issn.0366-6964.2025.02.021

摘要/Abstract

摘要：

旨在研究在冷冻稀释液中添加染料木素(genistein，GEN)对牛精液冷冻保存效果的影响，为优化冷冻稀释液，提高冷冻-解冻后牛精液品质提供理论依据。本研究采集6头年龄3~5岁、体重(586±20) kg、健康状况良好的秦川种公牛的新鲜精液，将检测合格的精液混合后用含不同浓度GEN的冷冻稀释液稀释后冷冻保存；根据冷冻稀释液中GEN添加浓度的不同，本试验分为5个组，0 μg·mL^-1 GEN组(对照组)、30 μg·mL^-1 GEN组、60 μg·mL^-1 GEN组、90 μg·mL^-1 GEN组、120 μg·mL^-1 GEN组，每组3个重复；解冻后利用牛全自动精子质量分析仪测定精子的运动性能，低渗肿胀检测法和花生凝集素荧光标记法检测精子顶体完整性和质膜完整性，试剂盒检测精子的线粒体膜电位(mitochondrial membrane potential，MMP)、抗氧化性能、丙二醛(malondialdehyde，MDA)含量和活性氧(reactive oxygen species，ROS)水平。结果，与对照组相比，30、60、90 μg·mL^-1 GEN组的前向运动精子率显著提升(P < 0.05)，30、60 μg·mL^-1 GEN组的精子的顶体完整性显著提高(P < 0.05)；60 μg·mL^-1 GEN组的ROS水平显著低于其余组(P < 0.05)；添加了GEN的各组与对照组相比，T-AOC和SOD含量均显著增加(P < 0.05)，MDA含量在30、60、90 μg·mL^-1 GEN组显著减少(P < 0.05)，GSH-px含量在60、90 μg·mL^-1 GEN组显著增加(P < 0.05)，CAT含量在60、120 μg·mL^-1 GEN组显著增加(P < 0.05)；60 μg·mL^-1 GEN组的MMP显著高于其余组(P < 0.05)。试验结果表明，冷冻稀释液中染料木素添加浓度为60 μg·mL^-1时对牛精液具有最佳的冷冻保存效果。添加染料木素可以提高牛精液中精子的抗氧化性能，抑制精子的氧化应激，缓解精子顶体和线粒体等细胞结构受到的冻融损伤，增强解冻后精子的运动性能，从而达到改善冷冻-解冻后牛精液品质的效果。

关键词: 染料木素, 牛精液冷冻保存, 线粒体膜电位, 抗氧化性能

Abstract:

The study aimed to investigate the effect of genistein (GEN) added to the semen extender on the cryopreservation of semen from cattle, and to provide a theoretical basis for optimizing the semen extender of cattle and improving the sperm quality of cattle after freezing and thawing. In this study, the fresh semen of 6 health Qinchuan bulls aged 3-5 years old, weighing (586±20) kg were collected, and the semen that passed the test was mixed, diluted with different concentrations of genistein for cryopreservation. According to the different concentrations of GEN in the semen extender, this study was divided into 5 groups, 0 μg·mL^-1 GEN group (control group), 30 μg·mL^-1 GEN group, 60 μg·mL^-1 GEN group, 90 μg·mL^-1 GEN group, and 120 μg·mL^-1 GEN group, with 3 replicates in each group. After thawing, bovine automatic sperm quality analyzer was used to measure the motility performance of sperm, hypotonic swelling detection method and peanut lectin fluorescent labeling method were used to detect sperm acrosome integrity and plasma membrane integrity, and the kits were used to detect mitochondrial membrane potential (MMP), antioxidant performance, malondialdehyde (MDA) content and reactive oxygen species (ROS) level. Compared with the control group, the forward motility sperm rate of the 30, 60 and 90 μg·mL^-1 GEN groups was significantly increased (P < 0.05), and the acrosome integrity of sperm in the 30 and 60 μg·mL^-1 GEN groups was significantly increased (P < 0.05). The ROS level in the 60 μg·mL^-1 GEN group was significantly lower than that in the other groups (P < 0.05). Compared with the control group, the contents of T-AOC and SOD were significantly increased in the GEN group (P < 0.05), the MDA content was significantly decreased in the 30, 60 and 90 μg·mL^-1 GEN groups (P < 0.05), and the GSH-px content was significantly increased in the 60, 90 μg·mL^-1 GEN groups (P < 0.05), the CAT content was significantly increased in the 60, 120 μg·mL^-1 GEN groups (P < 0.05). The MMP of the 60 μg·mL^-1 GEN group was significantly higher than that of the other groups (P < 0.05). The results showed that the supplementation of the semen extender with genistein could improve the antioxidant performance of sperm in bovine semen, inhibit the oxidative stress of sperm during cryopreservation, alleviate the freeze-thaw damage of sperm acrosomes and mitochondria, and improve the motility performance of sperm after thawing, so as to improve the quality of bull semen after freezing-thawing, and the concentration of genistein was 60 μg·mL^-1.

Key words: genistein, cryopreservation of bovine semen, mitochondrial membrane potential, antioxidant performance

中图分类号:

S823.3

梁恩堂, 李化轩, 陈帅成, 李果, 孙格格, 昝林森. 染料木素对牛精液冷冻保存效果的影响[J]. 畜牧兽医学报, 2025, 56(2): 700-710.

LIANG Entang, LI Huaxuan, CHEN Shuaicheng, LI Guo, SUN Gege, ZAN Linsen. Effect of Genistein on Semen Cryopreservation of Bull[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 700-710.

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项目 Item	染料木素浓度/(μg·mL^-1)GEN					P-value
项目 Item	0	30	60	90	120	Treat	Linear	Quadratic
活动精子率/% Motility	57.20±2.76	60.40±0.40	63.37±0.52	58.83±1.20	57.87±1.67	0.05	0.96	0.02
前向运动精子率/% Promotility	31.33±1.81^b	44.57±1.42^a	48.17±0.47^a	43.50±2.26^a	40.87±4.34^ab	< 0.01	0.04	< 0.01
前向运动精子平均速率/ (μm·s^-1) Velocity	33.33±1.76	38.00±0.58	39.00±0.58	38.33±0.88	36.67±1.86	0.26	0.11	0.01

项目 Item	染料木素浓度/(μg·mL^-1)GEN					P-value
项目 Item	0	30	60	90	120	Treat	Linear	Quadratic
质膜完整性/% Membrane integrity	57.20±1.16	60.06±0.56	63.28±1.35	58.39±1.83	57.93±1.89	0.18	0.98	0.02

项目 Item	染料木素浓度(μg·mL^-1) GEN					P-value
项目 Item	0	30	60	90	120	Treat	Linear	Quadratic
顶体完整性/% Acrosome integrity	54.61±0.61^c	59.23±0.81^b	64.58±0.42^a	57.63±0.48^bc	55.32±1.00^c	< 0.01	0.94	< 0.01

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