Improvement of sperm motility of Oncorhynchus mykiss and Salvelinus fontinalis by L-tryptophan.

Experiments were designed to test the influence of L-tryptophan supplementation of the extender on the sperm motility parameters and bacterial flora of brook trout (Salvelinus fontinalis) and rainbow trout (Oncorhynchus mykiss). The extender containing 0.3 M glucose in 10% Dimethyl sulfoxide (DMSO) was supplemented with 0, 0.5, 1, 2, and 4 mM L-tryptophan. Sperm samples stored for up to 12 days at 4°C for brook trout were motile while motility was not observed after six days of storage for rainbow trout. Diluted sperm samples were spread-plated onto Plate Count Agar (PCA) (Total Bacteria Count), Rose Bengal Agar (RBC) (Yeast-Mold Count), Violet Red Bile Dextrose (VRBD) (Enterobacteriaceae count) and Mannitol Salt Agar (MSA) (Micrococcus/Staphylococcus count) and counts were performed in duplicate and sampling occurred on day 0, 2, 4, 6, and 12 of cold storage. L-tryptophan supplementation of the extender yielded a positive effect, significantly prolonging sperm motility in S. fontinalis and O. mykiss compared to the control group (p < 0.05). The ideal results were obtained above a concentration of 0.5 mM for both species. Total bacteria count in fresh sperm (undiluted samples) was not detectable and not detected in sperm samples treated with different L-tryptophan concentrations. Yeast-mold, Enterobacteriaceae and Micrococcus/Staphylococcus were not detected from fresh and treated sperm brook trout and rainbow trout. On the basis of the results, L-tryptophan-based extender is useful for maintaining sperm quality in short-term storage. L-tryptophan had a negative effect on the bacterial flora. The results of the current study encourages further studies related to long-term storage and reproduction management. Abbreviations: DMSO: dimethyl sulfoxide; PCA: plate count agar; RBC: rose Bengal agar; VRBD: violet red bile dextrose; MSA: mannitol salt agar; SCA: sperm class analyzer; CFU: colony-forming unit.

CRYOPRESERVATION OF GOLDFISH (Carassius auratus) SPERMATOZOA: EFFECTS OF EXTENDER SUPPLEMENTED WITH TAURINE ON SPERM MOTILITY AND DNA DAMAGE.

BACKGROUND: Amino acids, present in seminal plasma at high concentration, protect spermatozoa against cell damage during cryopreservation. OBJECTIVE: Experiments were designed to analyze the effect of semen extender supplemented with taurine on post-thawed sperm motility and duration, as well as DNA damage. MATERIALS AND METHODS: Extenders were supplemented with 1, 2 or 4 mM taurine. Semen samples were diluted at the ratio of 1:9 with the extenders. Diluted samples were aspirated into 0.25 ml French straws and 0.1 ml pellets. DNA damage was assessed with the comet assay after cryopreservation. RESULTS: The percentage and duration of sperm motility were significantly increased by taurine. Additionally, sperm motility and the motility period in pellets were higher than in straws. The best concentration of taurine was 4 mM, and the highest post-thaw motility rate (72.50 ± 3.54 %) and duration (17.50 ± 0.71 s) were obtained from the extender with 4 mM in pellets. DNA damage was decreased after taurine supplementation. CONCLUSION: Pellets could be used for goldfish sperm cryopreservation. The addition of 4 mM taurine increases the post-thaw motility and decreases DNA damage on goldfish semen.

Effect of semen extender supplementation with cysteine on postthaw sperm quality, DNA damage, and fertilizing ability in the common carp (Cyprinus carpio).

Amino acids have an important biological role for prevention of cell damage during cryopreservation. The objective of this study is to determine the effects of cysteine on postthaw sperm motility, duration of sperm motility, DNA damage, and fertility in the common carp (Cyprinus carpio). Sperm collected from 10 individuals was cryopreserved in extenders containing different cysteine concentrations (2.5, 5, 10, and 20 mM). Semen samples diluted at the ratio of 1:9 by the extenders were subjected to cryopreservation. After dilution, the semen was aspirated into 0.25-mL straws; the straws were placed on the tray, frozen in nitrogen vapor, and plunged into liquid nitrogen. DNA damage was evaluated by comet assay after cryopreservation. Our results indicated that an increase in the concentration of cysteine caused a significant increase in the motility rate and duration of sperm in the common carp (C carpio; P < 0.05). Comparing all concentrations of cysteine, the best concentration of cysteine was 20 mM. Higher postthaw motility (76.00 ± 1.00%) and fertilization (97.00 ± 1.73%) rates were obtained with the extender at the concentration of 20 mM. Supplementation of the extender with cysteine was increased the fertilization and hatching rate and decreased DNA damage. Consequently, cysteine affected the motility, fertilization, and DNA damage positively, and extenders could be supplemented with cysteine.

Cryopreservation of rainbow trout Oncorhynchus mykiss spermatozoa: effects of extender supplemented with different antioxidants on sperm motility, velocity and fertility.

In present study, it was examined whether addition of different antioxidants to the cryopreservation extenders had an effect on semen post-thaw fertility and motility in rainbow trout (Oncorhynchus mykiss) and also it was investigated the sperm characteristics post-thaw sperm characteristics and fertility. The collected semen was pooled to minimize individual variation. Each pooled ejaculate was split into 12 equal aliquots and diluted with base extenders supplemented with the antioxidants, and a base extender with no additives (control). The pooled semen samples diluted at the ratio of 1:10 by the extenders were subjected to cryopreservation. Antioxidants were separately added to the extenders (one per experimental group): catalase (250 U/l), superoxide dismutase (250 U/l), peroxidase (250 U/l), oxidized glutathione (1.5 mmol/l), reduced glutathione (1.5 mmol/l), L-methionine (1.5 mmol/l), uric acid (0.25 mmol/l), L-ascorbic acid (0.5 mmol/l), α-tocopherol (2.0 mmol/l), ß-carotene (0.5 mmol/l) and carnitine (0.5 mmol/l). After dilution the semen was aspirated into 0.25 ml straws, the straws were placed on the tray, frozen for 10 min, and plunged into liquid nitrogen. Our results indicated that the post-thaw motility rate increased in extenders supplemented with uric acid, L-methionine, SOD, L-carnitine, α-tocopherol and L-reduced glutathione (p<0.05). The motility duration of frozen thawed semen increased in extenders supplemented with uric acid, L-methionine, SOD, α-tocopherol and L-reduced glutathione (p<0.05). Fertilization rate and hatching rate of frozen-thawed semen was not affected by the tested antioxidants. Consequently, the tested antioxidants affected the motility parameters and cryopreservation extenders could be supplement with antioxidants. This study suggested usage of antioxidants in the cryopreservation of rainbow trout.

Determination of bioaccumulation of heavy metals and selenium in tissues of brown trout Salmo trutta macrostigma (Duméril, 1858) from Munzur Stream, Tunceli, Turkey.

The objective of the present work was to determine the bioaccumulation of arsenic (As), cadmium (Cd), copper (Cu), lead (Pb), mercury (Hg), uranium (U) and selenium (Se) in gill, liver, and muscle tissues of the fresh water fish Salmo trutta macrostigma (Duméril, 1858) in Munzur Stream, Tunceli, Turkey. The highest concentrations of U (1.83 µg kg(-1)), Pb (119.84 µg kg(-1)) and Se (1.31 µg kg(-1)) were recorded in the gills of S. t. macrostigma. Concentrations of As (46.27 µg kg(-1)), Cd (109.19 µg kg(-1)), Hg (16.40 µg kg(-1)), Cu (18.19 µg kg(-1)) were recorded at highest levels in the liver. The results showed that there were significant differences in concentrations of As, Cd, Cu, Pb, Se, U and Hg in gill, liver and muscle tissue (p < 0.05). Heavy metals were within the edible parts of the investigated fish were in the permissible safety levels for human uses.