Skip to main content


Log in

Organic Zinc and Copper Supplementation–Associated Changes in Gene Expression and Protein Profiles in Buck Spermatozoa

Biological Trace Element Research Aims and scope Submit manuscript


Mineral supplementation has greater impact on male reproduction; however, the mechanism of action has not been studied in detail. The present study was aimed to deal with the lacuna in mechanism of action of mineral supplementation on improvement in sperm characteristics. A group of 40 bucks (aged 5 months) were assigned to 10 groups (4 in each group) based on their body weight and fed with concentrate mixture: basal roughage (minimal diet) in equal proportion to all the bucks. Among the 10 groups, one was considered as control, without any additional mineral supplementation, and the remaining 9 were treatment groups (3 groups each in Zn, Cu, and Zn + Cu). In treatment groups, organic Zn was fed in three different doses as 20, 40, and 60 mg/kg DM; organic Cu was fed in three different doses as 12.5, 25, and 37.5 mg/kg DM; and organic Zn + Cu was combinedly supplied as 20 + 12.5, 40 + 25, and 60 + 37.5 based on their mg/kg DM for 8 months period. The neat semen samples were processed for spermatozoal gene (stress- NOS3, HSP70, HIF1A; fertility- MTF1, MTA1, TIMP2, TNFa, and EGFR) expression studies through qRT-PCR and protein profile changes through single- and two-dimensional gel electrophoresis. Significantly, the stress-responsive genes were downregulated, and fertility-related genes were upregulated in treatment groups. A significant correlation had been noticed among the genes studied: HIF1A with MTA1 (P < 0.05) and MTF1 with EGFR, TIMP2, TNFa, and NOS3 (P < 0.01) respectively. The organic Zn and Cu feeding modulated the expression of stress- and fertility-related genes and protein abundance, thereby improved the sperm characteristics.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Data Availability



  1. Thornton PK (2010) Livestock production: recent trends, future prospects. Philos Trans R Soc Lond B Biol Sci 365:2853–2867.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Dagli NR, Bakshi SA, Gulawane SU, Baig MI, Vanage G, Deshmukh BT, Ghorpade PP (2014) Conception rate in Osmanabadi goats using frozen semen preserved in different cryoprotectants. Indian J Small Ruminants 20:101–103

    Google Scholar 

  3. Ranjhan SK (1998) Nutrient requirements of livestock and poultry. Indian Council of Agricultural Research, New Delhi

  4. Arangasamy A, Krishnaiah MV, Manohar N, Selvaraju S, Guvvala PR, Soren NM, Reddy IJ, Roy KS, Ravindra JP (2018) Advancement of puberty and enhancement of seminal characteristics by supplementation of trace minerals to bucks. Theriogenology 110:182–191.

    Article  CAS  PubMed  Google Scholar 

  5. Cheah Y, Yang W (2011) Functions of essential nutrition for high quality spermatogenesis. Adv Biosci Biotechnol 02(04):182–197.

    Article  CAS  Google Scholar 

  6. Van Niekerk FE, Van Niekerk CH (1989) The influence of experimentally induced copper deficiency on the fertility of rams. I. Semen parameters and peripheral plasma androgen concentration. J S Afr Vet Assoc 60(1):28–31

    PubMed  Google Scholar 

  7. Arangasamy A, Krishnaiah MV, Manohar N, Selvaraju S, Rani GP, Soren NM, Reddy IJ, Ravindra JP (2018) Cryoprotective role of organic Zn and Cu supplementation in goats (Capra hircus) diet. Cryobiology 81:117–124.

    Article  CAS  PubMed  Google Scholar 

  8. Hemalatha K, Arangasamy A, Selvaraju S, Krishnaiah MV, Rani GP, Mishra A, Soren NM, Reddy IJ, Ravindra JP (2018) Effect of dietary supplementation of organic zinc and copper on in vitro semen fertility in goat. Small Rumin Res 161:68–72.

    Article  Google Scholar 

  9. Narasimhaiah M, Arunachalam A, Sellappan S, Mayasula VK, Guvvala PR, Ghosh SK, Chandra V, Ghosh J, Kumar H (2018) Organic zinc and copper supplementation on antioxidant protective mechanism and their correlation with sperm functional characteristics in goats. Reprod Domest Anim 53(3):644–654.

    Article  CAS  PubMed  Google Scholar 

  10. Arangasamy A, Sharma RB, Hemalatha K, Krishnaiah MV, Selvaraju S, Rani GP, Binsila BK, Soren NM, Reddy IJ, Ravindra JP, Bhatta R (2018) Relationship of organic mineral supplementation and spermatozoa/white blood cells mRNA in goats. Anim Reprod Sci 197:296–304.

    Article  CAS  PubMed  Google Scholar 

  11. Krishnaiah M, Arangasamy A, Selvaraju S, Guvvala PR, Ramesh K (2019) Organic Zn and Cu interaction impact on sexual behaviour, semen characteristics, hormones and spermatozoal gene expression in bucks (Capra hircus). Theriogenology 130:130–139.

    Article  Google Scholar 

  12. Echeverry H, Yitbarek A, Munyaka P, Alizadeh M, Cleaver A, Camelo-Jaimes G, Wang P, Rodriguez-Lecompte JC (2016) Organic trace mineral supplementation enhances local and systemic innate immune responses and modulates oxidative stress in broiler chickens. Poult Sci 95(3):518–527.

    Article  CAS  PubMed  Google Scholar 

  13. Shan T, Dai P, Zhu P, Chen L, Wu W, Li Y, Li C (2017) Effect of an organic trace mineral premix on the semen quality, testicular morphology and gene expression related to testosterone synthesis of male broiler breeders. Rev Bras Ciência Avícola 19(3):481–488.

    Article  Google Scholar 

  14. Azpiazu R, Amaral A, Castillo J, Estanyol JM, Guimera M, Ballescà JL, Balasch J, Oliva R (2014) High-throughput sperm differential proteomics suggests that epigenetic alterations contribute to failed assisted reproduction. Hum Reprod 29(6):1225–1237.

    Article  CAS  PubMed  Google Scholar 

  15. Villemure M, Lazure C, Manjunath P (2003) Isolation and characterization of gelatin-binding proteins from goat seminal plasma. Reprod Biol Endocrinol 1:39.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Arangasamy A, Singh LP, Ahmed N, Ansari MR, Ram GC (2005) Isolation and characterization of heparin and gelatin binding buffalo seminal plasma proteins and their effect on cauda epididymal spermatozoa. Anim Reprod Sci 90(3–4):243–254.

    Article  CAS  PubMed  Google Scholar 

  17. Oliveira CH, Silva AM, Silva LM, van Tilburg MF, Fernandes CC, Velho AL, Moura AA, Moreno FB, Monteiro-Moreira AC, Moreira RA, Lima IM (2015) Growth, testis size, spermatogenesis, semen parameters and seminal plasma and sperm membrane protein profile during the reproductive development of male goats supplemented with de-oiled castor cake. Reprod Toxicol 53:152–161.

    Article  CAS  PubMed  Google Scholar 

  18. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative pcr and the 2−ΔΔCT method. Methods 25(4):402–408

    Article  CAS  PubMed  Google Scholar 

  19. Somashekar L, Selvaraju S, Parthipan S, Ravindra JP (2015) Profiling of sperm proteins and association of sperm PDC-109 with bull fertility. Syst Biol Reprod Med 61(6):376–387.

    Article  CAS  PubMed  Google Scholar 

  20. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685.

    Article  CAS  PubMed  Google Scholar 

  21. Gromova I, Celis J (2006) Protein detection in gels by silver staining: a procedure compatible with mass spectrometry. In: Celis J (ed) Cell biology. Academic Press, pp 219–223.

    Chapter  Google Scholar 

  22. Kurzawski M, Kaczmarek M, Kłysz M, Malinowski D, Kazienko A, Kurzawa R, Droździk M (2017) MMP2, MMP9 and TIMP2 polymorphisms affect sperm parameters but not fertility in Polish males. Andrologia 49(5):e12654.

    Article  CAS  Google Scholar 

  23. Belardin LB, Antoniassi MP, Camargo M, Intasqui P, Fraietta R, Bertolla RP (2019) Semen levels of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinases (TIMP) protein families members in men with high and low sperm DNA fragmentation. Sci Rep 9(1):903.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ma LI, Li W, Zhu HP, Li Z, Sun ZJ, Liu XP, Zhao J, Zhang JS, Zhang YQ (2010) Localization and androgen regulation of metastasis-associated protein 1 in mouse epididymis. PLoS ONE 5(11):e15439.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Heuchel R, Radtke F, Georgiev O, Stark G, Aguet M, Schaffner W (1994) The transcription factor MTF-1 is essential for basal and heavy metal-induced metallothionein gene expression. EMBO J 13(12):2870–2875

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kumar R, Wang RA (2016) Structure, expression and functions of MTA genes. Gene 582(2):112–121.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Andrews GK (2001) Cellular zinc sensors: MTF-1 regulation of gene expression. Biometals 14(3/4):223–237.

    Article  CAS  PubMed  Google Scholar 

  28. Selvaraj A (2005) Metal-responsive transcription factor (MTF-1) handles both extremes, copper load and copper starvation, by activating different genes. Genes Dev 19(8):891–896.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Wu W, Jaspers I, Zhang W, Graves LM, Samet JM (2002) Role of Ras in metal-induced EGF receptor signaling and NF-κB activation in human airway epithelial cells. Am J Physiol Cell Mol Physiol 282(5):L1040–L1048.

    Article  CAS  Google Scholar 

  30. Samet JM, Silbajoris R, Wu W, Graves LM (1999) Tyrosine phosphatases as targets in metal-induced signaling in human airway epithelial cells. Am J Respir Cell Mol Biol 21(3):357–364.

    Article  CAS  PubMed  Google Scholar 

  31. Chu A, Foster M, Hancock D, Bell-Anderson K, Petocz P, Samman S (2015) TNF-α gene expression is increased following zinc supplementation in type 2 diabetes mellitus. Genes Nutr 10(1):440.

    Article  CAS  PubMed  Google Scholar 

  32. Alexandrova A, Bandžuchová E, Kebis A, Kukan M, Kuba D (2007) Copper decreases gene expression of TNF-α, IL-10, and of matrix metalloproteinases MMP-2 and MMP-9 in isolated perfused rat livers. Biologia (Bratisl) 62(3):365–369.

    Article  CAS  Google Scholar 

  33. Erata GÖ, KoçakToker N, Durlanık Ö, Kadıoğlu A, Aktan G, AykaçToker G (2007) The role of heat shock protein 70 (Hsp 70) in male infertility: is it a line of defense against sperm DNA fragmentation? Fertil Steril 90(2):322–327.

    Article  CAS  PubMed  Google Scholar 

  34. Zhao W, Li Z, Ping P, Wang G, Yuan X, Sun F (2018) Outer dense fibers stabilize the axoneme to maintain sperm motility. J Cell Mol Med 22(3):1755–1768.

    Article  CAS  PubMed  Google Scholar 

  35. Hatayama T, Asai Y, Wakatsuki T, Kitamura T, Imahara H (2019) Regulation of hsp70 synthesis induced by cupric sulfate and zinc sulfate in thermotolerant HeLa cells. J Biochem 114(4):592–597

    Article  Google Scholar 

  36. Cortese-Krott MM, Kulakov L, Opländer C, Kolb-Bachofen V, Kröncke K-D, Suschek CV (2014) Zinc regulates iNOS-derived nitric oxide formation in endothelial cells. Redox Biol 2:945–954.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Yang SJ, Keen CL, Lanoue L, Rucker RB, Uriu-Adams JY (2007) Low nitric oxide: a key factor underlying copper-deficiency teratogenicity. Free Radic Biol Med 43(12):1639–1648.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Inoue N, Ikawa M, Isotani A, Okabe M (2005) The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs. Nature 434(7030):234–238.

    Article  CAS  PubMed  Google Scholar 

Download references


We acknowledge the Department of Biotechnology, Jain University, Bengaluru, India, for helping to register the doctoral program; CSIR HRDG (Council of Scientific & Industrial Research), Government of India, for providing the CSIR fellowship to do Ph.D; The Director, ICAR-NIANP, Bengaluru, for providing research workspace and facilities at the institute; and The Director, DBT, Government of India, for supporting the research work through chemicals and kits.


This research work was funded by DBT, Ministry of Science & Technology, Government of India (BT/PR10901/AAQ/1/581/2014) and CSIR HRDG, Government of India.

Author information

Authors and Affiliations



VM conducted the research and prepared the manuscript; PG helped in statistical analysis of the data. AA designed the experiment and helped for preparing the article; SS helped in designing the work; JG assisted in preparing and proof reading the manuscript.

Corresponding author

Correspondence to Arangasamy Arunachalam.

Ethics declarations

Ethics Approval

This study was carried out in the Reproductive Physiology Laboratory at ICAR-NIANP, Bengaluru, with necessary approval from institutional animal ethics committee (IAEC).

Consent to Participate


Consent for Publication


Conflict of Interest

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 209 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mayasula, V.K., Arunachalam, A., Sellappan, S. et al. Organic Zinc and Copper Supplementation–Associated Changes in Gene Expression and Protein Profiles in Buck Spermatozoa. Biol Trace Elem Res 200, 1626–1639 (2022).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: