Carboxymethylchitosan with medium-molecular-weight affects kinectics and acrosome of stallion sperm after freezing/thawing.

Semen cryopreservation ensures the storage of stallion genetics for an unlimited time. The improvement of extenders with new antioxidant substances can optimize the properties of post-thawed semen. The study aimed to investigate the addition effect of medium-molecular-weight carboxymethylchitosan (CQm) derivates to freezing diluent of stallion sperm after freezinf/thawing. Twice a week, five ejaculates of four stallions were obtained, totalizing 20 ejaculates. Semen was diluted in commercial freezing extender (Botucrio) supplemented with CQm: control (0), 0.75, 1.5, and 3 mg/mL. Samples were filled in straws (0.5 mL) and submitted to freezing and storage at -196°C. Thawing was performed at 37°C/30 s, and the samples of each group were analyzed for kinetics, plasma membrane integrity, acrosome membrane integrity, and mitochondrial membrane potential . The addition of 1.5 and 3 mg/mL CQm showed lower values (P < .05) of total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP) and wobble (WOB), comparing to control group. Besides, it was observed lower (P < .05) percentages of sperm with intact acrosomes in the group treated with 3 mg/mL of CQm than control group. In conclusion, high concentration of medium-molecular-weight carboxymethylchitosan to freezing diluent damages kinematic and acrosome of stallion sperm after freezing/thawing.

Effect of antifreeze protein type III on frozen/thawed of spermatozoa recover from goat epididymis.

The objective of this study was to evaluate the effect of antifreeze protein type III (AFP III) on the freezing of epididymal spermatozoa of goats. A total of 16 pairs of testicles were collected in a slaughterhouse and transported at approximately 5 °C in a thermal box. Epididymal spermatozoa were recovered by retrograde lavage and evaluated using a phase contrast microscope. Then, they were cryopreserved in extender based on Tris-egg yolk, supplemented with AFP III (0, 1, 10, 100 µg/mL), using an automated system. After thawing (37 °C/30 s), the spermatozoa kinetics were evaluated using the CASA automated system; and plasma and acrosome membrane integrity, mitochondrial membrane potential, and intracellular ROS production, by flow cytometry. There was no difference (P ≥ 0.05) between the experimental groups for the parameters of spermatozoa kinetics, mitochondrial membrane potential, and ROS production. However, the integrity of plasma and acrosome membranes of frozen spermatozoa with 100 µg/mL of AFP III was lower (P < 0.05) than the control group. It was concluded that the addition of AFP III to the Tris-egg yolk extender, used in the freezing of sperm obtained from the epididymis of goats, did not improve the preservation of these cells.

The interference of ozone gas in kinects and mitochondrial potential of equine sperm submitted on cryopreservation.

The objective of this study was to evaluate the effects of the addition of different concentrations of ozone to quarter horse semen submitted to cryopreservation. Six ejaculates from four stallions were collected and were divided in four experimental groups: a control group (BotuCRIO® extender) and three other groups with BotuCRIO® ozonized at concentrations of 6, 8 and 12 µg of O3/mL. The semen samples were diluted (200 x 106 spermatozoa/mL), filled in straws and frozen. After thawing (37 ºC, 30s), the samples were evaluated at 0, 30 and 60 minutes of incubation regarding sperm kinetics by a computer-assisted sperm analysis (CASA), and plasma membrane integrity (PMI), acrosome integrity (ACi) and mitochondrial membrane potential (MMP) by fluorescent probes. There was a reduction in the kinetic parameters total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) in all groups during the thermoresistance test (TT), a pattern also found in PMI and MMP analyses (p<0.05). There was no difference (p>0.05) between the control and treatment (6, 8, and 12 µg of O3/mL) groups, in any of the evaluated times for the kinetic parameters TM, linearity (LIN), straightness (STR), wobble index (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). Regarding the VCL, VSL and VAP parameters, the group treated with 6 µg did not differ from the control or from 8 µg, but was higher than 12 µg at 30 and 60 minutes. ACi and PMI did not differ between groups (p>0.05), but PMI was lower in groups 8 µg and 12 µg compared to the control and 6 µg (p<0.05). It was concluded that the addition of ozone does not present beneficial effects for cryopreservation of equine semen at the concentrations used and decreases important parameters of fertility.

Effect of green tea extract (Camellia sinensis) on the spermatic parameters of Wistar rats submitted or not to testicular heat shock.

The aim of this study was to evaluate the effect of green tea extract (GTE) on the spermatic parameters of Wistar rats, submitted or not to testicular heat shock (HS). For this, 48 animals were treated according to the experimental groups (G1: not exposed to HS and untreated; G2: exposed to HS and untreated; G3: not exposed to HS and treated with GTE; G4: exposed to HS and treated with GTE). Subgroups of rats were euthanized on days 15, 30, and 60 to recover the spermatozoa. The total motility (TM), vigor, spermatic morphology and concentration, mitochondrial membrane potential, plasma membrane integrity, and acrosome integrity (ACi) were analyzed. The TM was higher in G1 and G3 than in G2 and G4 on day 30, and higher in G4 on day 60. The overall means of TM and vigor were higher in G1 and G3 than in G2 and G4, as well as TM on day 60. For the morphology, G2 and G4 were lower than G1 and G3 on day 15, and G4 was lower than G1 and G3 on day 30. Moreover, in G1 and G3 morphology was higher on days 15 and 30, and in G4 it was lower on day 30, with the overall means being higher in G1 and G3 than in G2 and G4, as well as on days 15 and 60 compared to day 30. The overall mean of ACi, on day 30, was lower than on days 15 and 60 for all the groups. Therefore, HS is shown to be widely deleterious to the gametes, and the daily administration of 100 mg/kg green tea extract does not improve the spermatic parameters of Wistar rats, submitted or not to testicular HS, although it leads to better recovery of spermatic motility and morphology at 60 days.

Concentration of soybean lecithin affects short-term storage success of goat semen related with seminal plasma removal.

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.