l-Carnitine (LC) plays a key role in sperm metabolism, easily providing energy through b-oxidation, which positively affects motility. The objective of this study was to investigate the association between blood plasma and seminal plasma LC levels, as well as the effect of LC as an additive in a skimmed milk-based extender during sperm storage at 5°C. In the first experiment, semen and blood samples from 14 Quarter Horse stallions were used. The LC content in blood plasma and seminal plasma was determined by spectrophotometry and their relationships with seminal parameters were evaluated. In the second experiment, ejaculates (n = 16) from four Quarter Horses were used. Each ejaculate was split into four treatment groups with different LC concentrations: 0 (control), 0.5, 1.0, and 2.0 mM. Sperm motility, integrity of plasma and acrosomal membranes, intracellular reactive oxygen species content, and plasma membrane stability were evaluated immediately after samples reached 5°C (0 hour) and after 24, 48, and 72 hours. There was a positive correlation ( p < 0.05) between LC levels in seminal plasma with both sperm concentration and plasma and acrosomal membrane integrity. Furthermore, the addition of LC (1 and 2 mM) preserved the motility of equine sperm stored at 5°C. It was concluded that the concentrations of LC with seminal plasma present correlate to semen parameters and the addition of LC to skimmed milk-based extender preserves the motility of equine sperm stored at 5°C for up to 48 hours.
The addition of antioxidants to semen cryopreservation extenders has been employed for combating oxidative damage. This work aimed to evaluate the addition of carotenoid canthaxanthin to a cryopreservation extender of ram semen. Three breeder rams were used and, after semen collection, with 48-hour intervals between collection, the samples were included in the pool formation (n = 6). The experimental groups comprised 0 (control), 0.1, 1, 10, and 25 μM of canthaxanthin. After thawing (37°C/30 s) and incubation at 37°C for 2 hours, semen aliquots from each group were evaluated for sperm kinetics (CASA), the integrity of the plasma and acrosomal membranes (iPAM), intracellular reactive oxygen species (ROS) production, and lipid peroxidation (LPO) by flow cytometry associated with the image. The control group and canthaxanthin 1 μM after incubation at 37°C for 2 hours showed increases of curvilinear velocity and amplitude of lateral head displacement with decreases of linearity, straightness, and wobble (p < 0.05), which were not observed for the canthaxanthin 10 and 25 μM. The supplementation of a Tris-egg yolk extender with canthaxanthin had no effect on the iPAM, intracellular ROS production in viable spermatozoa, or LPO. In conclusion, supplementation with 10 and 25 μM of canthaxanthin in a Tris-egg yolk extender used for ram semen cryopreservation is able to protect ovine sperm from kinetic changes after incubation at 37°C for 2 hours post-thawing.
The objective of this study was to investigate the need of seminal plasma removal for short-term cooling of buck semen in soybean lecithin (SL) based extender. Each pool was divided equally, and one half was subjected to centrifugation to remove seminal plasma (SP-), while the other half remained with seminal plasma (SP+). Then, both SP+ and SP-samples were diluted in two SL extenders (extender A = 1% SL; extender B = 2% SL), cooled to 5ºC and stored for 48 hours. The sperm kinetics, evaluated by CASA, and plasma membrane integrity (PMI), acrosomal integrity (ACI) and high mitochondrial membrane potential (HMMP), evaluated by epifluorescence microscopy, were determined within five minutes after reaching 5°C (T0), as well as after 24 (T24) and 48 (T48) hours of storage. Interactions (seminal plasma vs. extender vs. time;) were observed for all variables assessed. Total and progressive motility and other variables of sperm kinetics decreased after 24 hours of cooling in the SP+ group, and after 48 hours of storage, these same variables were lower in SP+/B compared to SP-/B groups. Furthermore, SP+ reduced PMI (extender B, T48), HMMP (A and B extenders, T48) and ACI (extender A, T0) compared to SP-samples. The interactions between seminal plasma and soybean lecithin phospholipids seemed to occur in a time-dependent manner. It was concluded that the removal of seminal plasma improves the quality of goat semen that was cooled in a soybean lecithin-based extender, especially when using 2% soybean lecithin.
The objectives of this study were to evaluate goat sperm sorting in continuous Percoll® density gradients and gamete freezability, in the presence or absence of phenolic antioxidants. For this, semen pools were sorted, frozen, and evaluated. The non-selected group (NSg) presented lower progressive motility (PM), linearity (LIN), straightness (STR), and wobble (WOB) than the selected groups, and straight line velocity (VSL) compared to those with catechin or resveratrol. The amplitude of lateral head displacement (ALH) was higher in NSg, and quercetin reduced the mitochondrial membrane potential (MMP). After thawing, the NSg presented lower PM than the selected groups, VSL and VAP (average path velocity) than the selected group with or without catechin, LIN and WOB than the selected with or without catechin or resveratrol, and STR than the selected with catechin. Moreover, NSg presented higher ALH and BCF than the samples selected with or without catechin. Plasma membrane integrity and intact and living cells were higher in the selected groups, and MMP was lower in the NSg and the selected group with quercetin. Thus, centrifugation in Percoll® continuous density gradients is a viable methodology to select goat sperm compatible with the freezing, especially in the presence of catechin or resveratrol.
Estudo da vascularização folicular e do corpo lúteo de éguas cíclicas tratadas com extrato de pituitária equina utilizando ultrassom Doppler colorido
This study was conducted to evaluate the effects of lycopene on quality parameters of cryopreserved ram semen. Semen samples were collected from three Santa Inês rams to form a seminal pool (n = 8). Aliquots from each pool were diluted in Tris-egg yolk extender to create experimental groups containing 0 µM (control), 0.1 µM, 1 µM and 5 µM lycopene. The samples were evaluated for sperm kinetics, integrity of plasma and acrosomal membranes, intracellular reactive oxygen species (ROS) production and lipid peroxidation immediately after thawing and after incubation for two hours at 37 °C. The addition of lycopene had no effect on the parameters that were evaluated at the time of thawing when compared with the control. However, after incubation, the groups with added lycopene showed a decrease in progressive motility. All experimental groups showed a significant reduction in linearity and straightness following incubation. Furthermore, the 5 µM lycopene group showed a decrease in wobble and an increase in amplitude of lateral head displacement. In conclusion, the addition of lycopene to the freezing extender of ram semen affected the kinetics parameters of cryopreserved spermatozoa after a two-hour incubation period.
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