K. M. Lemes

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.

Uterine Vascular Perfusion and Involution During the Postpartum Period in Mares

ABSTRACT In horses, limited data is found regarding the vascular events during uterine involution at the puerperal period. Thus, we aimed to evaluate the morphological aspects (size of uterus and intrauterine fluid content) and the hemodynamics (endometrial and mesometrial vascular perfusion) of the uterus during its postpartum involution process. Ten mares were daily scanned by transrectal color Doppler ultrasonography from the first day postpartum (d1) to the 16th day after first postpartum ovulation (D0 = ovulation). The formerly gravid horn (GH) and formerly nongravid horn (NH) were individually evaluated. A reduction (P < .05) in the uterine diameter was observed during the first 7 days postpartum, and the rate of uterine involution decreased after this period. The involution was completed on d21 and d24 for the NH and GH, respectively. Presence of intrauterine fluid was present in large amounts between d1 and d2 postpartum, followed by a decrease (P < .05) between d4 and d7. No fluid was observed after d16 postpartum or after the third day postovulation (D3). During the early postpartum period, an increase (P < .05) in the endometrial and mesometrial vascularization was detected, respectively, between d1 and d4, and between d1 and d2. The vascular perfusion did not differ after d4 for endometrial tissue, whereas was reduced (P < .05) between d2 and d10 for mesometrium. After the first postpartum ovulation, an increase (P < .05) in vascular perfusion was observed from D0 to D5, followed by a decrease (P < .05) between D5 and D11 and an increase (P < .05) between D11 and D14. The novel vascular perfusion profile here described in the endometrium and mesometrium after ovulation is similar to the uterine vascular profile observed during estrous cycles and early pregnancy, indicating a fast return of the mares uterus to cycling postpartum conditions. HIGHLIGHTSImportant physiological changes involved in the uterine involution were described.Great reduction in the uterine diameter was observed during the first days postpartum.The uterine involution process was completed until 4 weeks postpartum.Uterine vascularity has a transitional increase during first 2 to 4 days postpartum.Uterine vascularity after ovulation is similar to that observed during early pregnancy.

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.