1. CINNAMTANNIN B-1 REDUCE ROS PRODUCTION IN RED DEER SPERMATOZOA
M.R. FERNÁNDEZ-SANTOSa*, F. SANCHEZ-RUBIOa, E. DEL OLMOa, M. INIESTAa, O. GARCÍA-ÁLVAREZa, A. MAROTO-MORALESa, M. RAMÓNb, P. JIMÉNEZ-RABADÁNa, L. ANEL-LÓPEZa, J.J. GARDEa , A.J. SOLERa.
aSaBio IREC (CSIC – UCLM)
bRegional Center of Animal Selection and Reproduction (CERSYRA) JCCM
(Faculty of Pharmacy, UCLM)
Introduction
Antioxidants could improve sperm media, extending the viability of spermatozoa. Cinnamtannin B-1 (CNB-1) is a naturally occurring A-type proanthocyanidin that was initially isolated from the bark of Cinnamomum zeylanicum and has also been found in a limited number of plants including Linderae umbellateae and L. nobilis. A number of studies has reported different cellular effects of CNB-1, which are mostly mediated by its antioxidant activity. Thus, we tested if the supplementation of incubation media with different concentrations of CNB-1 would protect thawed red deer epididymal spermatozoa against oxidative stress (spontaneous and induced).
Material and Methods
Epididymal sperm samples from six stags were frozen in Tris-citrate-fructose extender. Thawed sperm samples were pooled. The pool was washed free of freezing extender by diluting with three volumes of BGM-3, centrifuging (600g, 5 min, room temperature) and removing the supernatant. The pellet was resuspended in BGM-3 up to 30×106 cellsmL−1. The protective ability of CNB-1 was tested on red deer spermatozoa incubated at 37 °C. Sperm samples were incubated with 0, 0.1, 1, 10 and 100 μM of CNB-1, with or without oxidative stress (100μM Fe2+) and analyzed at 0, 2 h and 4 h after starting the incubation.
Sperm parameters evaluated
Acrosome status (NAR)
Viability (YO-PRO®-1/PI)
Free radical production (ROS production):CM-H2DCFDA/IP
NAR (%) evaluated for a 1:20 dilution in 2% glutaraldehyde, M cacodylate/HCl buffer (pH 7.3)
Flow cytometry
Statistical analysis (R statistical environment): To analyze the effects of time, antioxidant and oxidant we used linear mixed-effects models. We replicated the experiment 6 times.
Results and Discussion
Acrosome status
Viability
ROS production (CM-H2DCFDA) * *
Control
Normal Apical Ridge (%)
Control
Ros production (median)
Oxidant
Yo-PRO-1 -/ PI- (%)
Control
Oxidant
Oxidant ** **
Cinnamtannin B-1 (µM)
Cinnamtannin B-1 (µM)
Cinnamtannin B-1 (µM)
*P< 0.05, **P< 0.01
High concentrations of CNB-1 (100 μM) preserved acrosome status in oxidized samples after 4 h of incubation (P<0.05). Moreover, lower concentrations of CNB-1 showed limited ability to reduce ROS production although high concentrations (100 μM) lowered ROS production in oxidized samples after 2 h of incubation (P<0.01)) and 4 h (P<0.01). CNB-1 seems a promising new antioxidant, but its particular effects on sperm physiology must be further studied.
Acknowledgements
This work has been supported by the Spanish Ministry of Science and Innovation (Project AGL )