木犀草素对秦川牛精子冷冻保存的影响

doi:10.11843/j.issn.0366-6964.2025.07.018

摘要/Abstract

摘要：

旨在探究在稀释液中添加木犀草素(luteolin，LUT)对秦川牛精液冷冻保存效果的影响。本研究采集了6头年龄为3~5岁、体重为(586±20)kg且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，用添加不同浓度LUT的冷冻稀释液进行稀释，并进行冷冻保存。根据冷冻稀释液中LUT添加浓度的不同，本试验分为5个组：0 mg·mL^-1 LUT组(对照组)、0.02 mg·mL^-1 LUT组、0.04 mg·mL^-1 LUT组、0.08 mg·mL^-1 LUT组和0.1 mg·mL^-1 LUT组，每组设置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；采用精子低渗肿胀试验检测质膜完整性；使用荧光染色法检测精子顶体完整性和线粒体活率；并使用试剂盒检测精子中过氧化氢酶(catalase，CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase，GSH-Px)的活性以及丙二醛(malondialdehyde，MDA)含量的变化。与对照组相比，0.02 mg·mL^-1 LUT组和0.04 mg·mL^-1 LUT组的精子活力、直线运动速度均显著提高(P < 0.05)，质膜完整性和顶体完整性也显著提高(P < 0.05)。同时，0.02 mg·mL^-1 LUT组、0.04 mg·mL^-1 LUT组和0.08 mg·mL^-1 LUT组的精子线粒体活率、CAT和GSH-Px活性均显著提高(P < 0.05)，且各试验组的MDA含量均显著降低(P < 0.05)。LUT的最适添加量为0.02 mg·mL^-1。在稀释液中添加LUT可以显著提高牛精液中精子的抗氧化能力，保护精子结构的完整性，缓解冷冻-解冻过程对精子造成的损伤，进而增强解冻后精子的运动性能，从而有效提升冷冻精液的品质。

关键词: 秦川牛, 木犀草素, 精液, 冷冻保存

Abstract:

The aim of this experiment was to investigate the effect of adding luteolin (LUT) to the diluent on the cryopreservation effect of semen from Qinchuan cattle. In this study, fresh semen was collected from 6 Qinchuan breeding bulls aged 3-5 years, weighing (586±20) kg and in good health. The tested semen was mixed, diluted with freeze-diluent with different concentrations of LUT added, and frozen for preservation. According to the different concentrations of LUT added in the cryodilution solution, this experiment was divided into 5 groups: 0 mg·mL^-1 LUT group (control group), 0.02 mg·mL^-1 LUT group, 0.04 mg·mL^-1 LUT group, 0.08 mg·mL^-1 LUT group and 0.1 mg·mL^-1 LUT group, with 3 replicates set in each group. After thawing, sperm motility was measured using a bovine automatic sperm quality analyser; plasma membrane integrity was detected using the sperm hypotonic swelling test; sperm acrosome integrity and mitochondrial viability were detected using fluorescent staining; and spermatozoa were tested for catalase (CAT) activity, glutathione peroxidase (GSH-Px) activity, and malondialdehyde (MDA) content using a kit. Compared with the control group, sperm viability, linear motility speed, plasma membrane integrity and acrosome integrity were significantly increased in the 0.02 mg·mL^-1 LUT and 0.04 mg·mL^-1 LUT groups (P < 0.05). Meanwhile, sperm mitochondrial viability, CAT and GSH-Px activity in the 0.02 mg·mL^-1 LUT group, 0.04 mg·mL^-1 LUT group and 0.08 mg·mL^-1 LUT group were significantly increased (P < 0.05), and MDA content was significantly decreased (P < 0.05) in all experimental groups. The optimum amount of LUT added was 0.02 mg·mL^-1. The addition of LUT to the diluent could significantly improve the antioxidant capacity of spermatozoa in bovine semen, protect the structural integrity of spermatozoa, alleviate the damage to spermatozoa caused by the freezing-thawing process, and then enhance the motility of spermatozoa after thawing, thus effectively improving the quality of the frozen semen.

Key words: Qinchuan cattle, luteolin, semen, cryopreservation

中图分类号:

S823.3

吴斯林, 杨本顺, 叶苗苗, 梁恩堂, 李付强, 马伟东, 昝林森, 赵春平, 杨武才. 木犀草素对秦川牛精子冷冻保存的影响[J]. 畜牧兽医学报, 2025, 56(7): 3244-3251.

WU Silin, YANG Benshun, YE Miaomiao, LIANG Entang, LI Fuqiang, MA Weidong, ZAN Linsen, ZHAO Chunping, YANG Wucai. Effect of Luteolin on Semen Cryopreservation of Qinchuan Bull[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3244-3251.

图/表 8

表 1

图 1

表 2

图 2

表 3

图 3

表 4

表 5

参考文献 25

1	PINI T , MAKLOSKI R , MARUNIAK K , et al. Mitigating the effects of oxidative sperm dna damage[J]. Antioxidants (Basel), 2020, 9 (7): 589.
2	ZHAO J , JIN Y , DU M , et al. The effect of dietary grape pomace supplementation on epididymal sperm quality and testicular antioxidant ability in ram lambs[J]. Theriogenology, 2017, 97, 50- 56.
3	DAGHIGHKIA H , OLFATIKARAJI R , HOSEINKHANI A , et al. Effect of rosemary (Rosmarinus officinalis) extracts and glutathione antioxidants on bull semen quality after cryopreservation[J]. Span J Agric Res, 2014, 12 (1): 98- 105.
4	ALQAWASMEH O A , ZHAO M , CHAN C P , et al. Green tea extract as a cryoprotectant additive to preserve the motility and DNA integrity of human spermatozoa[J]. Asian J Androl, 2021, 23 (2): 150- 156.
5	TVRDÁ E , MICHALKO J , ÁRVAY J , et al. Characterization of the Omija (Schisandra chinensis) extract and its effects on the Bovine sperm vitality and oxidative profile during in vitro storage[J]. Evid Based Complement Alternat Med, 2020, 2020, 7123780.
6	ZOU Y , LUO X , FENG Y , et al. Luteolin prevents THP-1 macrophage pyroptosis by suppressing ROS production via Nrf2 activation[J]. Chem Biol Interact, 2021, 345, 109573.
7	RUNGSUNG S , SINGH T U , RABHA D J , et al. Luteolin attenuates acute lung injury in experimental mouse model of sepsis[J]. Cytokine, 2018, 110, 333- 343.
8	AKARSU S A , ACıSU T C , GÜNGÖR I H , et al. The effect of luteolin on spermatological parameters, apoptosis, oxidative stress rate in freezing rabbit semen[J]. Pol J Vet Sci, 2023, 26 (1): 91- 98.
9	何涛. 绿原酸、木犀草素和黄精多糖对猪精液冷冻保存效果的研究[D]. 兰州: 甘肃农业大学, 2021.
	HE T. Effects of chlorogenic acid, luteolin and polygonatum polysaccharides on cryopreservation of porcine semen[D]. Lanzhou: Gansu Agricultural University, 2021. (in Chinese)
10	张保平, 刘风华, 吴咏兰. 精子低渗肿胀试验在精液检测中的应用[J]. 内蒙古医学院学报, 1999, 1999 (3): 201- 202.
	ZHANG B P , LIU F H , WU Y L . The sperm hypotonic swelling test in semen detection[J]. Journal of Inner Mongolia Medical University, 1999, 1999 (3): 201- 202.
11	AMIRAT L , TAINTURIER D , JEANNEAU L , et al. Bull semen in vitro fertility after cryopreservation using egg yolk LDL: a comparison with Optidyl, a commercial egg yolk extender[J]. Theriogenology, 2004, 61 (5): 895- 907.
12	孙艳青, 李青旺, 耿果霞, 等. 葡萄籽原花青素对猪精液冷冻保存效果的研究[J]. 家畜生态学报, 2016, 37 (5): 46- 51.
	SUN Y Q , LI Q W , GENG G X , et al. Effects of grape seed proanthocyanidin on cryopreservation of boar spermatozoa[J]. Journal of Domestic Animal Ecology, 2016, 37 (5): 46- 51.
13	AITKEN R J , SMITH T B , LORD T , et al. On methods for the detection of reactive oxygen species generation by human spermatozoa: analysis of the cellular responses to catechol oestrogen, lipid aldehyde, menadione and arachidonic acid[J]. Andrology, 2013, 1 (2): 192- 205.
14	TSIKAS D . Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological samples: Analytical and biological challenges[J]. Anal Biochem, 2017, 524, 13- 30.
15	COLLODEL G , MORETTI E , MICHELI L , et al. Semen characteristics and malondialdehyde levels in men with different reproductive problems[J]. Andrology, 2015, 3 (2): 280- 286.
16	DARR C R , VARNER D D , TEAGUE S , et al. Lactate and pyruvate are major sources of energy for stallion sperm with dose effects on mitochondrial function, motility, and ros production[J]. Biol Reprod, 2016, 95 (2): 34.
17	GALLINA F G , GEREZ DE BURGOS N M , BURGOS C , et al. The lactate/pyruvate shuttle in spermatozoa: operation in vitro[J]. Arch Biochem Biophys, 1994, 308 (2): 515- 519.
18	SANTIANI A , EVANGELISTA S , SEPÚLVEDA N , et al. Addition of superoxide dismutase mimics during cooling process prevents oxidative stress and improves semen quality parameters in frozen/thawed ram spermatozoa[J]. Theriogenology, 2014, 82 (6): 884- 889.
19	LIEN E J , REN S , BUI H H , et al. Quantitative structure-activity relationship analysis of phenolic antioxidants[J]. Free Radic Biol Med, 1999, 26 (3-4): 285- 294.
20	HALLIWELL B , CROSS C E . Oxygen-derived species: their relation to human disease and environmental stress[J]. Environ Health Perspect, 1994, 102 (Suppl 10): 5- 12.
21	WU W , LI D , ZONG Y , et al. Luteolin inhibits inflammatory responses via p38/MK2/TTP-mediated mRNA stability[J]. Molecules, 2013, 18 (7): 8083- 8094.
22	XIA F , WANG C , JIN Y , et al. Luteolin protects HUVECs from TNF-α-induced oxidative stress and inflammation via its effects on the Nox4/ROS-NF-κB and MAPK pathways[J]. J Atheroscler Thromb, 2014, 21 (8): 768- 783.
23	PALHARES P C , ASSIS I D L , MACHADO G J , et al. Sperm characteristics, peroxidation lipid and antioxidant enzyme activity changes in milt of Brycon orbignyanus cryopreserved with melatonin in different freezing curves[J]. Theriogenology, 2021, 176, 18- 25.
24	ASHOKKUMAR P , SUDHANDIRAN G . Protective role of luteolin on the status of lipid peroxidation and antioxidant defense against azoxymethane-induced experimental colon carcinogenesis[J]. Biomed Pharmacother, 2008, 62 (9): 590- 597.
25	ROOBAN B N , SASIKALA V , GAYATHRI DEVI V , et al. Prevention of selenite induced oxidative stress and cataractogenesis by luteolin isolated from Vitex negundo[J]. Chem-Biol Interact, 2012, 196 (1-2): 30- 38.

木犀草素浓度/(mg·mL^-1) LUT concentration	精子活率/% Motility	精子活力/% Promotility	精子的前向运动平均速率/(μm·s^-1) Velocity
0	50.70±0.70^c	39.45±0.15^b	29.00±3.08^b
0.02	62.45±1.42^a	47.79±1.43^a	34.00±2.12^a
0.04	58.27±0.25^b	48.33±1.12^a	33.60±2.60^a
0.08	53.09±2.62^c	40.70±2.23^b	31.60±2.60^ab
0.1	52.55±2.75^c	40.45±2.20^b	32.20±1.09^ab

木犀草素浓度/(mg·mL^-1) LUT concentration	质膜完整性/% Plasma membrane integrity
0	50.23±0.85^c
0.02	58.91±0.58^a
0.04	56.35±1.95^b
0.08	52.07±0.64^c
0.1	50.55±0.59^c

木犀草素浓度/(mg·mL^-1) LUT concentration	顶体完整率/% Acrosome integrity
0	56.22±2.69^c
0.02	68.93±2.05^a
0.04	68.27±2.23^a
0.08	61.39±1.07^b
0.1	59.32±2.63^bc

木犀草素浓度/(mg·mL^-1) LUT concentration	线粒体活率/% Mitochondrial activity
0	48.08±0.13^d
0.02	56.23±1.11^a
0.04	53.78±0.51^b
0.08	51.93±0.48^c
0.1	49.13±0.50^d

木犀草素浓度/(mg·mL^-1) LUT concentration	过氧化氢酶活性 /(U·mL^-1) CAT	谷胱甘肽过氧化物酶活性/ (U·mL^-1) GSH-Px	丙二醛含量/ (mol·mL^-1) MDA
0	64.32±22.18^c	51.02±0.31^d	1.17±0.04^a
0.02	238.92±21.70^a	60.68±0.48^a	0.55±0.01^cd
0.04	154.40±16.03^b	59.54±0.94^ab	0.44±0.04^d
0.08	164.14±16.51^b	55.89±0.46^bc	0.67±0.09^c
0.1	74.18±20.01^c	52.88±5.95^cd	0.86±0.14^b