Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (14): 2850-2861.doi: 10.3864/j.issn.0578-1752.2022.14.014

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

Effect and Mechanism of Caffeic Acid Phenethyl Ester Alleviates Oxidative Stress in Liquid Preservation of Boar Semen Via the AMPK/FOXO3a Signaling Pathway

LAN Qun(),XIE YingYu,CAO JiaCheng,XUE LiE,CHEN DeJun,RAO YongYong,LIN RuiYi,FANG ShaoMing(),XIAO TianFang()   

  1. College of Animal Sciences Fujian Agriculture and Forestry University, Fuzhou 350002
  • Received:2021-05-16 Accepted:2021-06-21 Online:2022-07-16 Published:2022-07-26
  • Contact: ShaoMing FANG,TianFang XIAO;;


【Objective】The aim of this study was to explore the effect and mechanism of caffeic acid phenethyl ester (CAPE) relieves oxidative stress in boar semen through AMPK / FOXO3a signaling pathway. 【Method】 Eight adult (1-2 years) and healthy Landrace boars were selected to collect fresh semen, and the qualified samples were pooled for determination. Firstly, CAPE was added to basic diluent at the concentration of 0, 0.02, 0.04, 0.06, 0.08 and 0.10 g·L-1 at room temperature, respectively. To transferred the mixed specimens into electronic thermostat refrigerator for room temperature storage, semen samples were equilibrated at room temperature. The sperm kinematic parameters, including total motility and progressive motility, were determined by Computer-Aided Sperm Analysis (CASA) within 1-5 days, the best concentration of CAPE (0.06 g·L-1) was then selected. Secondly, the oxidative stress models were established by the ensure concentration of CAPE (0.06 g·L-1) and 400 μmol·L-1 hydrogen peroxide. At 1st, 3rd and 5th day, the total motility and progressive motility were measured by CASA, the plasma membrane integrity and acrosome integrity were evaluated by fluorescence probe technique, and the activities of catalase (CAT) and superoxide dismutase (SOD) were detected by antioxidant kit. After five days preservation, the mRNA expression of AMPK/FOXO3a signaling pathway related to genes, like AMPK, FOXO3a, SOD1, SOD2, CAT and apoptosis gene BAX, were determined by Real-time quantitative PCR (qPCR) technique, and the AMPK and p-AMPK protein expression were measured by western blot (WB). 【Result】(1) In the concentration screening experiment, 0.06 g·L-1 CAPE significantly improved the total motility on the 3rd day and maintained until the 5th day (P<0.05). Moreover, the progressive motility of sperm on the 1st day was significantly higher than other treatments (P<0.05). (2) In the oxidative stress experiment, the plasma membrane integrity, acrosome integrity, total motility, progressive motility, SOD and CAT activities of sperm in H2O2 group were significantly lower than blank group and CAPE+H2O2 group (P<0.05); however, there were no significant differences in total motility, acrosome integrity, CAT and SOD activities between the blank group and CAPE+H2O2 (P>0.05). Compared with CAPE+H2O2 group, the mRNA expression level of AMPK, FOXO3a, SOD1, SOD2, CAT in H2O2 group were significantly decreased (P<0.05), the expression level of BAX was significantly increased (P<0.05), while there was no significant difference in AMPK, SOD1, CAT and BAX between control group and CAPE+H2O2 group (P>0.05). In the expression level of proteins, AMPK, p-AMPK and p-AMPK/AMPK ratio were significantly higher in CAPE+H2O2 group in compared with H202 group (P<0.05). 【Conclusion】 The supplementation of 0.06 g·L-1 CAPE to basic diluent at room temperature could induce the transcription of downstream antioxidant molecules of AMPK/FOXO3a signaling pathway by promoting phosphorylation of AMPK expression, then alleviate the effect of H2O2 mediated oxidation which affected semen quality, and prolong its storage time. However, the in-depth molecular mechanism of CAPE protecting sperm fight with oxidative damage is still worth for further investigation.

Key words: boar semen, CAPE, room temperature preservation, semen quality, AMPK/FOXO3a signaling pathway

Fig. 1

Technical roadmap"

Fig. 2

Sperm plasma membrane integrity, acrosome integrity determination method"

Table 1

The primer sequences"

基因 Gene 引物序列 Primer sequence (5´-3´) 退火温度 Tm (℃) 产物长度 Length (bp) 基因编号 GenBank ID

Table 2

Effect of different concentrations of CAPE on total motility of boar sperm"

Preservation time (d)
咖啡酸苯乙酯 CAPE (g·L-1)
0 0.02 0.04 0.06 0.08 0.10
1 85.2±0.86 83.9±0.75 82.9±1.67 82.3±0.95 82.6±1.03 82.7±1.62
2 79.3±1.05 80.5±2.36 79.3±1.89 83.2±0.74 79.9±3.28 79.6±1.78
3 71.4±0.59c 74.0±1.41b 75.4±1.17b 80.0±1.40a 75.3±0.69b 74.0±0.40b
4 68.2±0.30d 70.3±0.71cd 71.1±1.07c 79.0±0.47a 75.4±1.44b 73.7±1.83b
5 62.1±1.20d 66.0±2.12c 68.6±1.24bc 79.3±0.68a 70.6±1.57b 67.5±1.51bc

Table 3

Effect of different concentrations of CAPE on progressive motility of boar sperm"

Preservation time (d)
咖啡酸苯乙酯CAPE (g·L-1)
0 0.02 0.04 0.06 0.08 0.10
1 61.3±1.88bc 61.9±0.91bc 60.4±1.82bc 62.9±0.16a 60.65±1.49bc 59.4±1.48c
2 50.8±0.44c 57.2±0.11b 58.6±1.44b 61.5±0.56a 56.7±1.22b 57.3±1.57b
3 49.4±2.10c 51.4±1.65bc 52.5±2.38bc 59.0±0.57a 54.9±1.50b 52.3±0.92bc
4 47.2±0.49c 49.8±0.95bc 52.4±1.02bc 58.5±0.82a 53.6±3.20ab 49.8±3.97bc
5 42.2±1.35c 42.7±1.39c 50.1±0.76b 56.0±0.47a 48.6±0.69b 41.7±1.69c

Fig. 3

Statistical analysis chart of motility parameters, structural integrities and antioxidant enzyme activities of spermatozoa preserved at room temperature A and B show the sperm total motility and progressive motility respectively; C and D represent the integrity of sperm plasma membrane and acrosome respectively; E and F denoted sperm CAT activity and SOD activity respectively. Different small letters mean significant difference among different treatment groups(P<0.05);Same small letters mean not significant difference among different treatment groups(P>0.05); CONT: Control group, H2O2: 40μmol·L-1 hydrogen peroxide, CAPE+H2O2: 0.06 g·L-1 phenethyl caffeic acid + 40μmol·L-1 hydrogen peroxide. The same as below"

Fig. 4

The mRNA expression levels of AMPK/FOXO3a signaling pathway related genes"

Fig. 5

Western blot analysis of AMPK and p-AMPK protein expression A: AMPK and p-AMPK protein expression bands; B: AMPK protein expression; C: p-AMPK protein expression; D: the ratio between p-AMPK and AMPK"

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