China Animal Husbandry and Veterinary Medicine ›› 2023, Vol. 50 ›› Issue (6): 2380-2387.doi: 10.16431/j.cnki.1671-7236.2023.06.022

• Genetics and Breeding • Previous Articles     Next Articles

Effects of Alpha-linolenic Acid on Buffalo Ovarian Granulosa Cells Cultured in vitro

XIAO Peng1,2,3, SHANG Jianghua1,2, YANG Chunyan1,2, LI Mengqi1,2, DUAN Anqin1,2, MA Xiaoya1,2, FENG Chao1,2, HUANG Chenqian1,2, ZHANG Bo1,2, ZHOU Jinchen1,2, WEI Kelong1,2, ZHENG Wei1,2, ZHENG Haiying1,2   

  1. 1. Guangxi Key Laboratory of Buffalo Genetics, Reproduction and Breeding, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning 530001, China;
    2. Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Nanning 530001, China;
    3. College of Animal Science and Technology, Guangxi Vocational University of Agriculture, Nanning 530004, China
  • Received:2023-01-28 Online:2023-06-05 Published:2023-05-30

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Abstract: 【Objective】 The experiment was to study the effect of α-linolenic acid(ALA) on cell viability, cell apoptosis, cell cycle-related gene expression and hormone secretion function of buffalo ovarian granulosa cells during in vitro culture.【Method】 In order to screen the optimal concentration of ALA for in vitro cultivation of buffalo ovarian granulosa cells, 1×104/mL of buffalo ovarian granulosa cells with an initial cell count were added to a 96 well plate.After 12 h of cell adhesion, the cells replaced the culture medium with 0 (control group), 10, 50, 100 and 200 μmol/L ALA and treated under the same conditions for 24 h, and cell viability was detected using CCK-8 to screen the optimal treatment concentration and use it for subsequent experiments.Subsequent experiments were divided into two groups:The control group (0 μmol/L) and the treatment group(50 μmol/L).Real-time quantitative PCR was used to detect the relative expression of B-cell lymphoma 2 (Bcl-2), cysteinyl aspartate-specific proteinase-3 (Caspase-3), cyclin-dependent kinase inhibitor 1 (p21), and proliferating cell nuclear antigen (PCNA) genes.Immunofluorescence was used to detect and quantitatively analyze the apoptosis related protein Caspase-3 and cell cycle related protein p21.ELISA was used to detect the levels of estradiol (E2) and progesterone (P4) secreted by buffalo granulosa cells in the culture medium.【Result】 Compared with the control group, the cell viability of buffalo ovarian granulosa cells increased with the increase of ALA concentration within the range of 10 to 50 μmol/L after 24 hours of ALA treatment, the cell viability was extremely significantly higher at a concentration of 50 μmol/L compared to the control group (P<0.01).However, as the concentration continued to increase, the activity of buffalo ovarian granulosa cells decreased, and at a concentration of 200 μmol/L, the cell viability was extremely significantly lower than that of the control group (P<0.01).Therefore, a concentration of 50 μmol/L ALA was selected for subsequent experiments.Compared to the control group, after 50 μmol/L ALA treatment, there was no significant difference in the relative expression of anti apoptotic gene Bcl-2 and proliferating cell nuclear antigen gene PCNA (P>0.05), but the expression of pro apoptotic gene Caspase-3 and cell cycle gene p21 were extremely significantly reduced at both mRNA and protein levels (P<0.01), while E2 and P4 levels were significantly or extremely significantly increased (P<0.05 or P<0.01).【Conclusion】 Adding 50 μmol/L ALA to the culture medium could enhance the proliferation activity, anti apoptotic ability, and hormone secretion ability of buffalo ovarian granulosa cells, and provided a theoretical reference for regulating the development of buffalo follicles.

Key words: α-linolenic acid(ALA); buffalo; granulosa cells; proliferation activity; gene expression

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