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| Affection of Azadirachtin on the Growth and Gene Expression of Pig (Sus scrofa) Testicular ST Cell Line |
| LU Sheng-Fei1, 2, RAN Xue-Qin1, *, NIU Xi1, HUANG Shi-Hui1, WANG Jia-Fu1, 2, *, LI Sheng1, DAI Xin-Lan1, MA Xin-Rui1 |
1 Institute of Life Science/College of Animal Science/Key laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang 550025, China;
2 Tongren University, Tongren 554300, China |
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Abstract As a natural insecticidal tetranortriterpenoid compound extracted from the seeds of the neem tree (Azadirachta indica), azadirachtin is currently widely used in agricultural products and vegetables for pest control. A large fraction of azadirachtin is lost and likely affects the environment and domestic animals by straw feeds exploitation. It has been reported that the intra-vas administration of neem oil resulted in the block of spermatogenesis in male rats. It is still unclear whether there is a role of azadirachtin on the function of pig testis. In order to gain insight into the importance of azadirachtin in male pig reproduction, this study was undertaken to investigate the effect of azadirachtin on testis in vitro by using pig testicular ST cell line. The status of ST cell growth was tracked under optical microscope and the proliferation or viability of cells were measured by 3- (4, 5-dimethyl thiazol-2-yl) -2, 5-diphenyl tetrazolium bromide (MTT) assay. Flow cytometry with annexin V FITC/ PI double staining was used to detect apoptosis of ST cell before and after azadirachtin treatment. The expression levels of four genes were determined by using quantitative real-time polymerase chain reaction (qRT-PCR) method, in which included MMP15 (matrix metallopeptidase 15), PICK1 (protein interacting with Cα kinase), BMP6 (bone morphogenetic protein 6), and MSH4 (MutS homolog 4). Using different dose of azadirachtin from 1 to 9 mg/L, the number of adherent cells was reduced, with cells rounded and suspended and appeared to be clustered under microscope. Based on determination of MTT assay, azadirachtin exhibited cytotoxic effects on the viability of ST cells in a dose and time-dependent manner with IC50 value of 7.646 mg/L at 24 h. Azadirachtin induced the apoptosis of ST cells in a dose- and time-dependent manner with a higher rate of early apoptotic cells at concentrations of 8 mg / L than that of 5 mg / L for 24 h of incubation (P<0.05). As the treatment concentration of azadirachtin changed from 5 mg/L to 8 mg/ L, the expression of all genes except MSH4 were rapidly decreased after dealting with azadirachtin at a dose-dependent way. The above findings demonstrated that azadirachtin had a cytotoxic impact on pig testis cell, and the induction of apoptosis might be one of the causes. It made a reasonable speculation that the function of pig testis was influenced by azadirachtin because the several gene expressions related with reproduction ability were disturbed intensely. It suggested that azadirachtin might inhibit the fertility of boar and need to have a mention on the detection and amount of azadirachtin residue in plant feedstuff in pig industry. The results could provide a basis for the residue control of azadirachtin in plant feedstuff.
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Received: 31 August 2018
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Corresponding Authors:
xqran@gzu.edu.cn; jfwang@gzu.edu.cn
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