Effects of taurine, cysteine and melatonin as antioxidant supplements to the freezing medium of Prochilodus brevis sperm.

Cryopreservation consist of a set of methods to preserve cells and tissues by drastically reducing the temperature. Among some undesired effects, cryopreservation might generate reactive oxygen species that lead to an increase of oxidative stress, causing damage to cells. This study aimed to test taurine, cysteine, and melatonin on the freezing of Prochilodus brevis sperm and assess its effects on post-thawed sperm quality. Sperm was collected and seven pools were formed (n = 7). They were diluted (1:9) in standard medium (5% glucose, 10% dimethyl sulfoxide and 5% egg yolk) supplemented or not (control) with taurine (0.3, 1.0, 3.16 or 10.0 mM), cysteine (0.3, 1.0, 3.16 or 10.0 mM) or melatonin (0.6, 1.12, 2.0 or 3.56 mM). Post-thawed sperm was evaluated for kinetic (total motility, velocities, and percentage of rapid cells), morphology and membrane and DNA integrity. Differences were found when melatonin was used as an antioxidant. For the variables rapid sperm and sperm velocities, 3.56 mM melatonin presented higher results than the control (melatonin 0 mM). Melatonin 2 mM was similar to 3.56 mM on rapid sperm, average path velocity (VAP) and curvilinear velocity (VCL). No difference was found between concentration 0 mM (control) and taurine treatments. As for cysteine, 0.3 mM presented the best results for rapid sperm than 10 mM, and higher VCL and VAP than 1 mM. Melatonin 3.56 mM presented higher results on kinetic parameters (rapid motility, VCL, VSL and VAP) than other tested antioxidants. Therefore, melatonin 3.56 mM is recommended to be added to the sperm freezing medium of P. brevis.

Sperm cryopreservation of Prochilodus brevis using different concentrations of non-permeable cryoprotectants.

The action of substances with non-permeable cryoprotectant potential, besides glucose, has not yet been studied for the species Prochilodus brevis. The objective of this work was to evaluate the action of four non-permeable cryoprotectants on this species sperm cryopreservation. Five pools were cryopreserved in a solution of 5% glucose and 10% dimethyl sulfoxide (Me2SO) associated or not (control) with cryoprotectants egg yolk (5, 10 or 12%), soy lecithin (2.5, 7.5 or 10%), sucrose (5, 10 or 20%) and lactose (5, 8 or 15%). After thawing, samples were evaluated for sperm kinetics (total motility, motility duration, velocities, and wobble - WOB), morphology and membrane and DNA integrity. The treatments containing egg yolk improved significantly (P<0.05) results when compared the control for the membrane integrity parameter. When compared to other treatments, egg yolk, at any concentration, presented higher results (P<0.05) for membrane integrity, total motility, curvilinear velocity (VCL) and average path velocity (VAP) parameters. Egg yolk also showed the best results for WOB, but it did not differ from 5% and 8% lactose and 5% and 20% sucrose. Soy lecithin had the lowest percentages of morphologically normal sperm (P<0.05), while the other treatments did not differ from each other. There was no difference regarding DNA integrity data. Thus, 5% egg yolk is indicated as a non-permeable cryoprotectant for P. brevis, in association with 5% glucose and 10% Me2SO.

Determination of the ideal volume of activating solution and the optimal ratio of spermatozoa per oocyte for Prochilodus brevis fertilization.

Prochilodus brevis is a rheophilic species with a threatened natural population that promotes studies aimed at optimizing reproduction in captivity. The correct quantity of inseminating dose and activating solution volume significantly improves fertilization rates, thereby increasing productivity in captivity. The objective of this study was to determine the proportion of sperm per oocyte and the ideal volume of activating solution to be used in the assisted fertilization of P. brevis. Gametes were collected and fertilization performed in two steps. In step 1, the ideal proportion of spermatozoa was determined based on the fertilization rate:oocyte by testing six doses of semen: D1 = 30 × 103, D2 = 150 × 103, D3 = 300 × 103, D4 = 3 × 106, D5 = 5 × 106, and D6 = 10 × 106. In step 2, the fertilization and hatching rates were evaluated using different volumes of activating solution (V1 - 25 ml, V2 - 50 ml, V3 - 75 ml,V4 - 100 ml, V5 - 125 ml, and V6 - 150 ml). A linear regression equation was estimated from steps 1 and 2. The Student-Newman-Keuls test was used to compare the means. In step 1, the percentage of fertilization increased linearly, reaching a plateau of 51.69%. In step 2, the best fertilization rates were obtained with an estimated ideal volume of 75.64 ml per 2 ml of oocytes. Therefore, the proportion of 928,410.29 sperm:oocyte, associated with the volume of 75.64 ml of water per 2 ml of oocytes, provided the maximum reproductive performance for P. brevis.