Skip to main content Accessibility help

Effects of in ovo injection of vitamin C on heat shock protein and metabolic genes expression

  • Y. F. Zhu (a1), M. B. Bodinga (a1), J. H. Zhou (a1), L. Q. Zhu (a1), Y. L. Cao (a1), Z. Z. Ren (a1) and X. J. Yang (a1)...


Some studies have shown that the excessive metabolic heat production is the primary cause for dead chicken embryos during late embryonic development. Increasing heat shock protein (HSP) expression and adjusting metabolism are important ways to maintain body homeostasis under heat stress. This study was conducted to investigate the effects of in ovo injection (IOI) of vitamin C (VC) at embryonic age 11th day (E11) on HSP and metabolic genes expression. A total of 320 breeder eggs were randomly divided into normal saline and VC injection groups. We detected plasma VC content and rectal temperature at chick’s age 1st day, and the mRNA levels of HSP and metabolic genes in embryonic livers at E14, 16 and 18, analysed the promoter methylation levels of differentially expressed genes and predicted transcription factors at the promoter regions. The results showed that IOI of VC significantly increased plasma VC content and decreased rectal temperature (P < 0.05). In ovo injection of VC significantly increased heat shock protein 60 (HSP60) and pyruvate dehydrogenase kinase 4 (PDK4) genes expression at E16 and PDK4 and secreted frizzled related protein 1 (SFRP1) at E18 (P < 0.05). At E16, IOI of VC significantly decreased the methylation levels of total CpG sites and −336 CpG site in HSP60 promoter and −1137 CpG site in PDK4 promoter (P < 0.05). Potential binding sites for nuclear factor-1 were found around −389 and −336 CpG sites in HSP60 promoter and potential binding site for specificity protein 1 was found around −1137 CpG site in PDK4 promoter. Our results suggested that IOI of VC increased HSP60, PDK4 and SFRP1 genes expression at E16 and 18, which may be associated with the demethylation in gene promoters. Whether IOI of VC could improve hatchability needs to be further verified by setting uninjection group.


Corresponding author


Hide All

These authors contributed equally to this work.



Hide All
Alamdari, N, Constantin-Teodosiu, D, Murton, AJ, Gardiner, SM, Bennett, T, Layfield, R and Greenhaff, PL 2010. Temporal changes in the involvement of pyruvate dehydrogenase complex in muscle lactate accumulation during lipopolysaccharide infusion in rats. Journal of Physiology-London 586, 17671775.10.1113/jphysiol.2007.149625
Bowker-Kinley, MM, Davis, WI, Wu, PF, Harris, RA and Popov, KM 1998. Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex. Biochemical Journal 329, 191196.
Chen, XY, Wei, PP, Xu, SY, Geng, ZY and Jiang, RS 2013. Rectal temperature as an indicator for heat tolerance in chickens. Animal Science Journal 84, 737739.10.1111/asj.12064
Cheng, WL, Qi, YQ, Li, T, Bing, W, Huang, WH and Chen, YJ 2017. Dicer promotes tumorigenesis by translocating to nucleus to promote SFRP1 promoter methylation in cholangiocarcinoma cells. Cell Death & Disease 8, e2628. doi: 10.1038/cddis.2017.57.
Chung, TL, Brena, RM, Kolle, G, Grimmond, SM, Berman, BP, Laird, PW, Pera, MF and Wolvetang, EJ 2010. Vitamin C promotes widespread yet specific DNA demethylation of the epigenome in human embryonic stem cells. Stem Cells 28, 18481855.10.1002/stem.493
Eskander, RN, Ali, S, Dellinger, T, Lankes, HA, Randall, LM, Ramirez, NC, Monk, BJ, Walker, JL, Eisenhauer, E and Hoang, BH 2016. Expression patterns of the WNT pathway inhibitors Dickkopf 3 and secreted frizzled-related proteins 1 and 4 in endometrial endometrioid adenocarcinoma: an NRG oncology/gynecologic oncology group study. International Journal of Gynecological Cancer 27, 181192.
Favomi, A, Ayo, JO, Akinpelu, AO and Nwanta, JA 2008. Effects of ascorbic acid on diurnal variations in rectal temperature of piglets during the hot-dry season. Nigerian Veterinary Journal 29, 23962403.
Gabriel, JE, Ferro, JA, Stefani, RMP, Ferro, MI, Gomes, SL and Macari, M 1996. Effect of acute heat stress on heat shock protein 70 messenger RNA and on heat shock protein expression in the liver of broilers. British Poultry Science 37, 443449.10.1080/00071669608417875
Gauger, KJ, Bassa, LM, Henchey, EM, Wyman, J, Bentley, B, Brown, M, Shimono, A and Schneider, SS 2013. Mice deficient in Sfrp1 exhibit increased adiposity, dysregulated glucose metabolism, and enhanced macrophage infiltration. PLoS One 8, e78320. doi: 10.1371/journal.pone.0078320.
Gorzowski, JJ, Eckerley, CA, Halgren, RG, Mangurten, AB and Phillips, B 1995. Methylation-associated transcriptional silencing of the major histocompatibility complex-linked hsp70 genes in mouse cell lines. Journal of Biological Chemistry 270, 2694026949.10.1074/jbc.270.45.26940
Janke, O, Tzschentke, B and Boerjan, M 2004. Comparative investigations of heat production and body temperature in embryos of modern chicken breeds. Avian and Poultry Biology Reviews 15, 191196.
Jastrebski, SF, Lamont, SJ and Schmidt, CJ 2017. Chicken hepatic response to chronic heat stress using integrated transcriptome and metabolome analysis. PLoS One 12, e0181900. doi: 10.1371/journal.pone.0181900.
Kadonaga, JT, Courey, AJ, Ladika, J and Tjian, R 1988. Distinct regions of Sp1 modulate DNA binding and transcriptional activation. Science 242, 15661570.10.1126/science.3059495
Kadonaga, JT, Jones, KA and Tjian, R 1986. Promoter-specific activation of RNA polymerase II transcription by Sp1. Trends in Biochemical Sciences 11, 2023.10.1016/0968-0004(86)90226-4
Kirchner, H, Nylen, C, Laber, S, Barrès, R, Yan, J, Krook, A, Zierath, JR and Näslund, E 2014. Altered promoter methylation of PDK4, IL-1B, IL-6, and TNF after Roux-en Y gastric bypass. Surgery for Obesity Related Diseases 10, 671678.10.1016/j.soard.2013.12.019
Kuroda, A, Rauch, TA, Todorov, I, Ku, HT, Al-Abdullah, IH, Kandeel, F, Mullen, Y, Pfeifer, GP and Ferreri, K 2009. Insulin gene expression is regulated by DNA methylation. PLoS One 4, e6953. doi: 10.1371/journal.pone.0006953.
Li, LC and Dahiya, R 2002. MethPrimer: designing primers for methylation PCRs. Bioinformatics 18, 14271431.10.1093/bioinformatics/18.11.1427
Li, SZ, Zhu, YF, Zhi, LH, Han, XY, Shen, J, Liu, YL, Yao, JH and Yang, XJ 2016. DNA methylation variation trends during the embryonic development of chicken. PLoS One 11, e0159230. doi: 10.1371/journal.pone.0159230.
Livak, KJ and Schmittgen, TD 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (T)(-Delta Delta C) method. Methods 25, 402408.
Moran, ET 2007. Nutrition of the developing embryo and hatchling. Poultry Science 86, 10431049.10.1093/ps/86.5.1043
Moura, CS, Lollo, PCB, Morato, PN and Amaya-Farfan, J 2018. Dietary nutrients and bioactive substances modulate heat shock protein (HSP) expression: a review. Nutrients 10, 683. doi: 10.3390/nu10060683.
Nagata, K, Guggenheimer, RA, Enomoto, T, Lichy, JH and Hurwitz, J 1982. Adenovirus DNA replication in vitro: identification of a host factor that stimulates synthesis of the preterminal protein-dCMP complex. Proceedings of the National Academy of Sciences of the United States of America 79, 64386442.10.1073/pnas.79.21.6438
Oliveira, JDE, Uni, Z and Ferket, PR 2008. Important metabolic pathways in poultry embryos prior to hatch. World’s Poultry Science Journal 64, 488499.
Purohit, GK, Mahanty, A, Suar, M, Sharma, AP, Mohanty, BP and Mohanty, S 2015. Investigating hsp gene expression in liver of Channa striatus under heat stress for understanding the upper thermal acclimation. Biomed Research International 2014, 110. doi: 10.1155/2014/381719.
Raghow, R, Yellaturu, C, Deng, X, Park, EA and Elam, MB 2008. SREBPs: the crossroads of physiological and pathological lipid homeostasis. Trends in Endocrinology and Metabolism 19, 6573.
Ritchie, S 2018. Platinum plan: a focus on prevention for optimal health and performance. Poultry Science Association 107th Annual Meeting, 23–26 July 2018, San Antonio, Texas, USA, pp. 229.
Sato, M, Tachibana, T and Furuse, M 2006. Heat production and lipid metabolism in broiler and layer chickens during embryonic development. Comparative Biochemistry and Physiology A-Molecular & Integrative Physiology 143, 382388.
Schmitt, E, Gehrmann, M, Brunet, M, Multhoff, G and Garrido, C 2007. Intracellular and extracellular functions of heat shock proteins: repercussions in cancer therapy. Journal of Leukocyte Biology 81, 1527.
Tullett, SG 1990. Science and the art of incubation. Poultry Science 69, 115.
Uni, Z, Yadgary, L and Yair, R 2012. Nutritional limitations during poultry embryonic development. Journal of Applied Poultry Research 21, 175184.
Xu, H, Uno, JK, Inouye, M, Collins, JF and Ghishan, FK 2005. NF-1 transcriptional factor(s) is required for basal promoter activation of the human intestinal NaPi-IIb cotransporter gene. American Journal of Physiology-Gastrointestinal and Liver Physiology 288, 175181.10.1152/ajpgi.00396.2004
Young, JI, Zuchner, S and Wang, GF 2015. Regulation of the epigenome by vitamin C. Annual Review of Nutrition 35, 545564.10.1146/annurev-nutr-071714-034228
Yu, JM and Bao, ED 2008. Effect of acute heat stress on heat shock protein 70 and its corresponding mRNA expression in the heart, liver, and kidney of broilers. Asian-Australasian Journal of Animal Science 21, 11161126.10.5713/ajas.2008.70703
Yuriko, K and Masaru, k 2006. WNT antagonist, SFRP1, is Hedgehog signaling target. International Journal of Molecular Medicine 17, 171175.
Zhang, M, Zhao, YJ, Li, Z and Wang, CY 2017. Pyruvate dehydrogenase kinase 4 mediates lipogenesis and contributes to the pathogenesis of nonalcoholic steatohepatitis. Biochemical and Biophysical Research Communications 495, 582586.
Zhao, YJ, Xu, SY, Jiang, RS and Qu, LJ 2012. Expression analysis of SFRP1 and PDK4 gene in livers of chicken under heat stress. China Poultry 34, 1215. doi: 10.3969/j.issn.1004-6364.2012.15.003.
Zhu, YF, Li, SZ, Sun, QZ and Yang, XJ 2019. Effect of in ovo feeding of vitamin C on antioxidation and immune function of broiler chickens. Animal doi: 10.1017/S1751731118003531.


Related content

Powered by UNSILO

Effects of in ovo injection of vitamin C on heat shock protein and metabolic genes expression

  • Y. F. Zhu (a1), M. B. Bodinga (a1), J. H. Zhou (a1), L. Q. Zhu (a1), Y. L. Cao (a1), Z. Z. Ren (a1) and X. J. Yang (a1)...


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed.