Original Research Does Coenzyme Q10 Exert Antioxidant Effect on Frozen Equine Sperm? Monique de Albuquerque Lagares a, * , Grazielle Caroline da Silva b , Steyner Franca Cortes b , Sabrina Barros Luz a , Auana Chaves de Resende a , Natalia de Castro Alves a , Raphael Rocha Wenceslau a , Rubens Stahlberg c a Departamento de Clínica e Cirurgia Veterin arias da Escola de Veterin aria da Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil b Departamento de Farmacologia do Instituto de Ci^ encias Biol ogicas da UFMG, Belo Horizonte, MG, Brazil c Faculdade de Medicina Veterin aria da Pontifícia Universidade Cat olica- PUC Minas, Betim, MG, Brazil article info Article history: Received 19 November 2019 Received in revised form 3 February 2020 Accepted 5 February 2020 Available online 13 February 2020 Keywords: Semen Horse Stallion Spermatozoa Ubiquinone CoQ10 abstract During semen cryopreservation, the sensitivity of equine sperm to oxidative stress is increased by the eliminated seminal plasma. Thus, antioxidant addition to the semen extender can be helpful to the sperm survival after freezing and thawing. This work aimed to test whether coenzyme Q10 (CoQ10) added in different concentrations to the INRA 82 freezing extender has antioxidant function on equine sperm to improve its fertilizing ability. Semen samples from five stallions were frozen with the extenders: (T1) INRA 82, control, (T2) T1þ 5 mM CoQ10, (T3) T1þ 25 mM CoQ10, and (T4) T1þ 50 mM CoQ10. After sample thawing, sperm motility and kinetics characteristics were evaluated using a computer-assisted sperm analysis and sperm membrane functionality and integrity were evaluated with a hypo-osmotic swelling test and an epifluorescence microscopy, respectively. The nitrite (NO 2 - ) and hydrogen peroxide (H 2 O 2 ) concentrations of the semen samples were measured with spectrophotometry. There was no difference on the sperm characteristics among all treatments (P > .05). However, the 25 mM CoQ10 (T3) decreased NO 2  concentration (6.7 ± 2.2 mM/mg protein) compared with the treatments T1, T2, and T4 (64.3 ± 3.7, 59.4 ± 5.3, 45.1 ± 8.6 mM/mg protein), respectively, as well H 2 O 2 concentration (1.8 ± 0.3 mM/mg protein) compared with the control (4.6 ± 0.4 mM/mg protein) and 5 mM CoQ10 treatments (4.8 ± 0.2 mM/mg protein, P < .05). In conclusion, 25 mM CoQ10 plays a significant role as antioxidant to the frozen equine sperm, decreasing NO 2  and H 2 O 2 concentrations. Thus, its addition to the INRA 82 freezing extender may be beneficial to the fertilizing ability of equine semen. © 2020 Elsevier Inc. All rights reserved. 1. Introduction The use of frozen semen has increased in the routine of equine artificial insemination. However, its fertility is still lower than fresh and cooled semen [1]. The semen cryopreservation results in changes in the sperm membranes, which leads to cell injury or death [2]. Furthermore, the decrease of the proportion of sperm surviving after semen freezing and thawing can be due to the addition and removal of cryoprotectant in molar proportions. This applies a considerable osmotic stress to the plasma membrane of sperm, decreasing its functional integrity [3]. During cryopreser- vation, osmotic stress induces reactive oxygen species (ROS) gen- eration by changes in cell volume resulting from the movement of water when solutes cross the sperm plasma membrane [2]. Sperm and seminal plasma under normal conditions neutralize the detrimental effects of ROS with antioxidant systems preventing internal cellular damage [4]. However, to avoid an in vitro prema- ture sperm capacitation by seminal plasma proteins [5], most of the equine seminal plasma is discarded after semen centrifugation during the freezing process [6]. Thus, sperm are extremely vulnerable to oxidative stress caused by the unbalance between Animal welfare/ethical statement: All experimental procedures were performed according to the Brazilian ethical and animal welfare principles for the utilization and care of animals used in research and were approved by the ethical Committee “Comiss~ ao de  Etica no Uso de Animais” (CEUA) at the Federal University of Minas Gerais (UFMG). Conflict of interest statement: The authors declare no conflicts of interest. * Corresponding author at: Monique de Albuquerque Lagares, Departamento de Clínica e Cirurgia Veterin arias da Escola de Veterin aria da Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil. E-mail address: mlagares@ufmg.br (M. de Albuquerque Lagares). Contents lists available at ScienceDirect Journal of Equine Veterinary Science journal homepage: www.j-evs.com https://doi.org/10.1016/j.jevs.2020.102964 0737-0806/© 2020 Elsevier Inc. All rights reserved. Journal of Equine Veterinary Science 88 (2020) 102964