Is Mitochondrial Uncoupler Dinitrophenol Effective in Counterbalancing the Dilution Effect in Buffalo Semen?

Authors

  • Venkateshappa Arjun Department of Gynaecology and Obstetrics, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125001, Haryana, India.
  • Pradeep Kumar Division of Animal Physiology and Reproduction, ICAR- Central Institute for Research on Buffaloes, Hisar-125001, Haryana, India.
  • Ravi Dutt Department of Gynaecology and Obstetrics, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125001, Haryana, India.
  • Dharmendra Kumar Division of Animal Physiology and Reproduction, ICAR- Central Institute for Research on Buffaloes, Hisar-125001, Haryana, India.
  • Amit Kumar Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141012, Punjab, India.
  • Renu Bala Division of Animal Physiology and Reproduction, ICAR- Central Institute for Research on Buffaloes, Hisar-125001, Haryana, India.
  • Nisha Verma Division of Animal Physiology and Reproduction, ICAR- Central Institute for Research on Buffaloes, Hisar-125001, Haryana, India.
  • Andonissamy Jerome Division of Animal Physiology and Reproduction, ICAR- Central Institute for Research on Buffaloes, Hisar-125001, Haryana, India.
  • Meenakshi Virmani Department of Veterinary Physiology and Biochemistry, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125001, Haryana, India.
  • Chandrashekhar Patil Department of Animal Genetics and Breeding, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125001, Haryana, India.
  • Shivani Bhardwaj Division of Animal Physiology and Reproduction, ICAR- Central Institute for Research on Buffaloes, Hisar-125001, Haryana, India.
  • Prem Singh Yadav Division of Animal Physiology and Reproduction, ICAR- Central Institute for Research on Buffaloes, Hisar-125001, Haryana, India.

DOI:

https://doi.org/10.48165/ijar.2022.43.1.4

Keywords:

Buffalo, CASA, Semen, Dinitrophenol, HOST, Mitochondrial membrane potential

Abstract

 Semen ejaculates were collected from Murrah buffalo (Bubalus bubalis) bulls and were diluted with low density lipoprotein (LDL) based extender into 20 million sperm/0.25 mL and 2 million sperm/0.25 mL. Dinitrophenol (DNP) was added (@ 0, 1, 10 and 50 µM) to 20 million and 2 million sperm concentration cryopreserved. After thawing, the parameters studied were plasma membrane integrity by HOST, sperm motility and kinetics by CASA, the thermal resistance of sperm by incubation test, mitochondrial superoxide status by MitoSOX through flow cytometry, mitochondrial membrane potential (MMP) evaluation by JC-1 through flow cytometry. There was no significant (P>0.05) change in plasma membrane integrity, sperm motility and kinematics; thermal resistance of sperm, mitochondrial superoxide status and hMMP in comparison to control within the 2 and 20 million sperm doses. Two million sperm doses resulted in low plasma membrane integrity, low thermal resistance of sperm, high mitochondrial superoxide status and decreased high MeMP and no significant (P>0.05) change in sperm motility and kinematics,  but reduced total motility, beat cross frequency  and no change in progressive motility, straight linear velocity, average path velocity, curvilinear velocity, the amplitude of lateral head displacement, straightness, linearity, and wobble in comparison to 20 million sperm doses. Supplementation of DNP (0, 1, 10 and 50 μM) in extender failed to improve semen quality in both 2 and 20 million sperm doses in buffalo. 

References

Arjun, V, Kumar, P., Dutt, R., Kumar, A., Bala, R., Verma, N., Jerome A., Virmani, M., Patil, C., Singh, S. and Kumar, D. (2021). "Is addition or removal of seminal plasma able to compensate for the dilution effect of buffalo semen?" Andrologia. e14123.

Arjun, V., Kumar, P., Dutt, R., Kumar, A., Bala, R., Verma, N., Jerome, A., Virmani, M., Patil, C. S., Bhardwaj, S., Kumar, D. and Yadav, P. S. (2022). Effect of mitochondria-targeted antioxidant on the regulation of the mitochondrial function of sperm during cryopreservation. Andrologia, e14431.

Ashworth, P.J.C., Harrison, R.A.P., Miller, N.G., Aplummer, J.M. and Watson, P.F. (1994). Survival of ram spermatozoa at high dilution: protective effect of simple constituents of culture media as compared with seminal plasma. Reprod. Fert. Dev., 6: 173-180.

Brand, M.D., Affourtit, C., Esteves, T.C., Green, K., Lambert, A.J., Miwa, S. and Parker, N. (2004). Mitochondrial superoxide: production, biological effects, and activation of uncoupling proteins. Free Radic. Biol. Med., 37(6): 755-767.

Centurion, F., Vazquez, J.M., Calvete, J.J., Roca, J., Sanz, L., Parrilla, I. and Martinez, E.A. (2003). Influence of porcine sperm adhesins on the susceptibility of boar spermatozoa to high dilution. Biol. Reprod., 69(2): 640-646.

Cunha, F.M., Caldeira da Silva, C.C., Cerqueira, F.M. and Kowaltowski, A.J. (2011). Mild mitochondrial uncoupling as a therapeutic strategy. Curr. Drug Targets., 12(6): 783-789.

Dalal, J., Chandolia, R.K., Pawaria, S., Kumar., A., Kumar, D., Selokar, N.L., Jerome A., . Yadav, P.S. and Kumar, P. (2020). Low-density lipoproteins protect sperm during

cryopreservation in buffalo: Unravelling mechanism of action. Mol. Reprod. Dev., 87(12):1231-1244.

Davila, M.P., Muñoz, P.M., Bolanos, J.M., Stout, T.A., Gadella, B.M., Tapia, J.A. and Pena, F.J. (2016). Mitochondrial ATP is required for the maintenance of membrane integrity in stallion spermatozoa, whereas motility requires both glycolysis and oxidative phosphorylation. Reproduction., 152(6): 683-694.

Dong, Q., Tollner, T.L., Rodenburg, S.E., Hill, D.L. and Vande Voort, C.A. (2010). Antioxidants, Oxyrase, and mitochondrial uncoupler 2, 4-dinitrophenol improved post-thaw survival of Rhesus monkey sperm from ejaculates with low cryosurvival. Fertil. Steril., 94(6): 2359-2361.

Fang, L., Bai, C., Chen, Y., Dai, J., Xiang, Y., Ji, X. and Dong, Q. (2014). Inhibition of ROS production through mitochondria-targeted antioxidant and mitochondrial uncoupling increases post-thaw sperm viability in yellow catfish. Cryobiology., 69(3): 386-393.

Ghaleno, L.R., Valojerdi, M.R., Janzamin, E., Chehrazi, M., Sharbatoghli, M. and Yazdi, R.S. (2014). Evaluation of conventional semen parameters, intracellular reactive oxygen species, DNA fragmentation and dysfunction of mitochondrial membrane potential after semen preparation techniques: a flow cytometric study. Arch. Gynecol. Obstet., 289(1): 173-180.

Gonzalez-Fernandez, L., Morrell, J.M., Pena, F.J. and Macias-Garcia, B. (2012). Osmotic shock induces structural damage on equine spermatozoa plasmalemma and mitochondria. Theriogenology., 78: 415–422.

Green, M. P., Harvey, A. J., Spate, L. D., Kimura, K., Thompson, J. G., Roberts, R. M. (2016). The effects of 2, 4-dinitrophenol and d-glucose concentration on the development, sex ratio, and interferon-tau (IFNT) production of bovine blastocysts. Molecul. Reprod. Dev., 83(1): 50-60.

Gruber, J., Fong, S., Chen, C.B., Yoong, S., Pastorin, G., Schaffer, S. and Halliwell, B. (2013). Mitochondria-targeted antioxidants and metabolic modulators as pharmacological interventions to slow ageing. Biotechnol. Adv., 31(5): 563-592.

Hansford, R. G., Hogue, B. A. and Mildaziene, V. (1997) Dependence of H2O2 formation by rat heart mitochondria on substrate availability and donor age. J. Bioenerg. Biomembr., 29: 89-95.

Kumar, A., Ghosh, S. K., Katiyar, R., Gemeda, A.E., Rautela, R., Bisla, A., Srivastava, N., Bhure, S. K., Devi, H.L. and Chandra, V. (2022). Supplementation of Mito TEMPO and acetovanillone in semen extender improves freezability of buffalo spermatozoa. Andrology, 10(4): 775-788.

Kumar, A., Ghosh, S.K., Katiyar, R., Rautela, R., Bisla, A., Ngou, A.A., Pande, M., Srivastava, N. and Bhure, S.K. (2021). Effect of Mito-TEMPO incorporated semen extender on physico-morphological attributes and functional membrane integrity of frozen thawed buffalo spermatozoa. Cryoletters, 42(2): 111-119.

Kumar, P., Saini, M., Kumar, D., Balhara, A.K., Yadav, S.P., Singh, P. and Yadav, P.S. (2015). Liposome-based semen extender is suitable alternative to egg yolk-based extender for cryopreservation of buffalo (Bubalus bubalis) semen. Anim. Reprod. Sci., 159: 38-45.

Kurland, C. and Andersson, S. (2000). Origin and evolution of the mitochondrial proteome. Microbiol. Molecul. Biol. Reviews., 64: 786-820.

Lambert, A.J. and Brand, M.D. (2004). Superoxide production by NADH: ubiquinone oxidoreductase (complex I) depends on the pH gradient across the mitochondrial inner membrane. Biochem. J., 382(2): 511-517.

Masoudi, R., Sharafi, M., Shahneh, A.Z., Towhidi, A., Kohram, H., Esmaeili, V., Shahverdi, A. and Davachi, N.D. (2016). Fertility and flow cytometry study of frozen-thawed sperm in cryopreservation medium supplemented with soybean lecithin. Cryobiology., 73(1): 69-72.

Maxwell, W.M.C. and Johnson, L.A. (1999). Physiology of spermatozoa at high dilution rates: The influence of seminal plasma. Theriogenology., 52(8): 1353-1362.

Mortimer, S. T. and Swan, M. A. (1995). Andrology: Kinematics of capacitating human spermatozoa analysed at 60 Hz. Human Reprod., 10(4): 873-879.

Nazari, M., Daghigh Kia, H., Ebrahimi, M., Najafi, A. and Mahdipour, M. (2020). Effect of targeted antioxidant 2, 4 dinitrophenol on improving qualitative and quantitative parameters of Ghezel ram sperm after freeze-thawing process during non-breeding season. Anim. Sci. J. (Pajouhesh & Sazandegi)., 130: 181-190.

Patil, S., Kumar, P., Singh, G., Bala, R., Jerome, A., Patil, C.S. and Sharma, R.K. (2020). ‘Semen dilution effect’ on sperm variables and conception rate in buffalo. Anim. Reprod. Sci., 214: 106304.

Perumal, P., Srivastava, S. K., Ghosh, S. K. and Baruah, K. K. (2014). Computer-assisted sperm analysis of freezable and non-freezable Mithun (Bos frontalis) semen. J. Anim., 675031: 1-6.

Silva, E.F., Junior, A.S.V., Cardoso, T.F., Stefanello, F.M., Kalb, A.C., Martínez, P.E. and Corcini, C.D. (2016). Reproductive toxicology of 2, 4 dinitrophenol in boar sperm. Toxicol. In Vitro., 35: 31-35.

Downloads

Published

2022-05-06

Issue

Vol. 43 No. 1 (2022): The Indian Journal of Animal Reproduction (Jan-Jun)

Section

Research Article

How to Cite

Arjun, V., Kumar, P., Dutt, R., Kumar, D., Kumar, A., Bala, R., … Yadav, P.S. (2022). Is Mitochondrial Uncoupler Dinitrophenol Effective in Counterbalancing the Dilution Effect in Buffalo Semen? . The Indian Journal of Animal Reproduction, 43(1), 22–29. https://doi.org/10.48165/ijar.2022.43.1.4
Crossref
Scopus
Google Scholar
Europe PMC