Renata Lançoni

An Efficient Technique to Detect Sperm Reactive Oxygen Species: The CellRox Deep Red ® Fluorescent Probe

Heat stress and testicular traumas increase sperm Reactive Oxygen Species (ROS) resulting in Oxidative Stress (OS) and injury of sperm. The fluorescent probe CellROX Deep Red® is not already used to detect ROS in spermatozoa. On this way, this study aims to evaluate its efficiency in detecting ROS in ram sperm. For that, it was used ejaculates from rams performing the in vitro induction of sperm OS in T0, T50 and T100. T0 was semen sample that was not submitted to OS induction, T50 was 50% induced to OS induction and T100 was entire submitted to OS induction. The production of ROS was detected by CellROX Deep Red®. Data polynomial regression analysis was performed and the result of determination coefficient was 0.728. Besides, sixteen rams were submitted to scrotal insulation during 72 hours performing the in vivo induction of OS sperm. Analyses of sperm motility, morphology, DNA fragmentation and ROS (detected by CellROX Deep Red®) were done two times before the scrotal insulation and two times afterwards. Data analysis of variance was performed from the periods (before x after) and it is observed that after scrotal insulation the total and progressive motility decrease (p<0.05) and total and major sperm defects and sperm DNA fragmentation increase (p<0.05). Moreover, after insulation, it was observed increase (p<0.05) in sperm showing moderate and intense OS. Thus, it is possible to conclude that CellROX® fluorescent probe is able to detect ROS production in ram sperm by in vitro and in vivo induction being a new technique to detect sperm OS.

Addition of Antioxidants Myoinositol, Ferulic Acid, and Melatonin and Their Effects on Sperm Motility, Membrane Integrity, and Reactive Oxygen Species Production in Cooled Equine Semen

Abstract The present study aimed to evaluate the influence of myoinositol (MYO), ferulic acid (FA), and melatonin (MEL) in equine cooled semen. Ejaculates were collected and distributed into the following four treatments: MYO, FA, MEL, and control. A skim milk–based extender was used. Samples were cooled at 5°C and evaluated at 0, 4, and 8 hours after storage for motility, plasma and acrosomal membranes integrity, mitochondrial potential, and production of reactive oxygen species (ROS). Motility characteristics were not affected by treatment, except for the amplitude of lateral head displacement, which was higher in MYO (8.3 ± 0.2) compared with the control group (7.8 ± 0.2). No difference was observed among treatments for intact plasma membrane (%). However, the percentage of cells with intact plasma and acrosomal membranes and high mitochondrial potential was greater in the MEL (78.1 ± 2.0) and FA groups (78.8 ± 1.7) compared with the control group (73.8 ± 2.0). The high mitochondrial potential (%) was also greater in groups treated with MEL (80.1 ± 1.9) and FA (81.0 ± 1.5) compared with the control group (76.6 ± 2.0). In addition, percentage of cells with intact acrosome membrane was greater in MEL group (99.7 ± 0.1) compared with all other treatments. ROS production was not affected by treatments. In conclusion, FA and MEL provided the best protection to mitochondria, acrosome, and plasma membranes, suggesting that the addition of these antioxidants to equine semen extender can improve sperm quality. HighlightsFerulic acid and melatonin provided the best protection to all membranes.Melatonin increases the percentage of cells with intact acrosome.No difference was observed among treatments for intact plasma membrane (%).Motility characteristics were not affected, except for the amplitude of lateral head displacement (higher in myoinositol).Reactive oxygen species production was not affected by treatments.

Validation of the CellRox Deep Red® fluorescent probe to oxidative stress assessment in equine spermatozoa

Considering the importance of ROS influence on sperm functionality and some limitations in sperm oxidative stress assessment methods, a field to studies of new techniques are still open. In this sense, the aim of this study is to validate the ROS detection technique through the CellRox Deep Red Reagent® probe in stallion sperm. Four stallions were used and the analyses were conducted on four replicates of semen samples from each of stallion (n = 16). The results of the polynomial regression presented a quadratic effect, high determination coefficient value (R2 = 0.88) and high significant P value (P < 0.0001). The CellRox Deep Red® fluorescent probe is able to detect reactive oxygen species in equine sperm, indicating accurately the occurrence of oxidative stress in stallion semen.

Melatonin Added to Cryopreservation Extenders Improves the Mitochondrial Membrane Potential of Postthawed Equine Sperm

&NA; Reactive oxygen species (ROS) can induce sperm damage, especially after cryopreservation. Melatonin (MEL) is a potent amphiphilic antioxidant, allowing free penetration to any compartment of cell. Ferulic acid (FA) is a phenolic compound exhibiting a wide range of beneficial effects against several pathologies due to its antioxidant property. This study aimed to evaluate the effect of MEL and FA on sperm quality of cryopreserved equine semen. Five ejaculates from four stallions were collected. For each ejaculate, the control group (conventional freezing extender BotuCrio; Botupharma, Botucatu, SP, Brazil) and two different concentrations of each antioxidant (MEL 2 mM, MEL 1 &mgr;M, FA 0.5 mM, and FA 1.2 mM) were tested. Sperm kinetics were assessed by CASA system (SCA). Integrity of plasma and acrosomal membranes and mitochondrial potential were assessed using fluorescent probes PI, Hoechst 33342, FITC‐PSA, and JC‐1. Sperm ROS production was evaluated by CellRox Deep Red. Comparisons among treatments were analyzed by generalized linear model (PROC GLM in SAS, version 9.3), and differences were compared with Duncans test (a value of P ≤ .05 was considered significant). MEL 1 &mgr;M treatment demonstrated higher percentage of sperm cells presenting intact plasma membrane, intact acrosome, and high mitochondrial membrane potential, which was due to higher percentage of sperm cells with high mitochondrial membrane potential. No differences among treatments were observed on ROS evaluation. It was concluded that semen treatment with 1 &mgr;M MEL improves mitochondrial function of equine sperm cells submitted to cryopreservation process. HighlightsMelatonin (MEL) had a better effect on cryopreserved equine sperm when compared with control and ferulic acid treatments.MEL improves sperm membrane integrity in the equine sperm cryopreservation process.MEL showed a direct effect on sperm mitochondria.