David Gumbao

Supplementation of the dilution medium after thawing with reduced glutathione improves function and the in vitro fertilizing ability of frozen‐thawed bull spermatozoa

In this study, we evaluated the effects of glutathione (l-gamma-glutamyl-l-cysteinylglycine; GSH) supplementation of the thawing extender on bull semen parameters to compensate for the decrease in GSH content observed during sperm freezing. To address these questions fully, we used a set of functional sperm tests. These included tests of sperm motility assayed by computer-assisted semen analysis, membrane lipid packing disorder, spontaneous acrosome reaction, free radical production [reactive oxygen species (ROS) generation], sperm chromatin condensation, DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling and acridine orange staining measured by flow cytometry. Finally, the in vitro penetrability of in vitro matured oocytes and the in vitro production of embryos were evaluated. The main findings emerging from this study were that addition of GSH to the thawing medium resulted in: (i) a higher number of non-capacitated viable spermatozoa; (ii) a reduction in ROS generation; (iii) lower chromatin condensation; (iv) lower DNA fragmentation; (v) higher oocyte penetration rate in vitro and (vi) higher in vitro embryo production compared with control group. Nevertheless, GSH had no significant effect on motion parameters or the occurrence of the spontaneous acrosome reaction. Addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen bull spermatozoa.

Supplementation of the thawing medium with reduced glutathione improves function of frozen-thawed goat spermatozoa

Sperm cryopreservation represents a useful tool in the management of reproduction in goat production. However, freezing and thawing produce physical and chemical stress on the sperm membrane that reduces their viability and fertilizing ability. In this study, firstly we evaluated the effects of reduced glutathione (GSH, 1 and 5mM) supplementation of the thawing extender on parameters of frozen-thawed goat spermatozoa. We used a set of functional sperm tests that included sperm motility assayed by computer-assisted semen analysis (CASA), membrane lipid packing disorder, spontaneous acrosome reaction, free radical production (ROS generation) and sperm chromatin condensation. The main findings from this study were that addition of GSH to the thawing medium resulted in: (1) a higher motility and progressive motility; (2) a higher number of non-capacitated viable spermatozoa; (3) higher number of viable spermatozoa with intact acrosome; (4) a reduction in ROS generation and (5) lower chromatin condensation. In a second study, the additions of reduced (GSH, 5mM) or oxidized glutathione (GSSG, 2.5mM) to the thawing media were evaluated. We confirmed the protective effect of GSH on the sperm functionality. The addition of GSSG to the thawing media was less protective to sperm functions compared to GSH. Addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen goat spermatozoa. The information derived from this study suggests the importance of oxidative stress as responsible for cryo-injury to spermatozoa and opens new windows to explore the practical application of antioxidants to improve the quality of post-thaw goat semen.

Assessment of two thawing processes of cryopreserved human sperm in pellets

In this study, we evaluated the effects of the thawing methodology on sperm function after cryopreservation in pellets. We compared the use of two thawing procedures: method (1) maintaining pellet for 10 min in air at room temperature, then another 10-min period in air at 37°C followed by dilution in a thawing medium; and method (2) immersing the pellets directly in thawing medium at 37°C for 20 min. This procedure leads to a higher rate of temperature increase and a dilution of the glycerol present in the freezing medium. We analyzed the effect of the thawing procedure on sperm motility, viability, membrane lipid packing disorder, acrosome status, reactive oxygen species (ROS) level and sperm chromatin condensation. This study revealed a positive effect of the M2 thawing methodology on sperm parameters. The percentage of spermatozoa with fast-linear movement is increased (M1: 17.26% vs. M2: 28.05%, p<0.01), with higher viability (M1: 37.81% vs. M2: 40.15%, p<0.01) and less acrosome damage (M1: 40.44% vs. M2: 35.45%, p=0.02). We also detected an increase in the percentage of viable spermatozoa with low membrane lipid disorder (M1: 31.36% vs. M2: 33.17%, p=0.03) and a reduction in chromatin condensation (44.62 vs. 46.62 arbitrary units, p=0.02). Further studies will be necessary to evaluate the possible clinical applications.