Cryopreservation of sea bass (Dicmcranchus labm~) spermatozoa in experimental and production simtiting conditions Christian Fauvel (I*), Marc Suquet (*) Catherine Dreanno (2, 3) , Vincenzo ionno (4), Bruno Menu cl) (') Station exptinwntale d'aquaculture, Ijwner, Cheme'n-de-Maguelone, 34250 Pabvas-ks-F!ots, France.

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In the present study we used two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to verify whether the protein expression of sea bass sperm was affected by the cryopreservation procedure. The protein profiles differed between fresh and frozen-thawed semen as revealed by visual inspection and by image analysis software. We identified 163 spots in fresh sperm; among these, 13 were significantly decreased and 8 were absent in two-dimensional gel obtained with cryopreserved sperm. Five of these spots were analyzed with MALDI-TOF, but only three showed a significant match in the databases used in bio-informatics analysis (PeptIdent, Mascot, and MS-Fit). In particular, spot 5 showed homology with a novel protein of zebrafish (similar to SKB1 of human and mouse), spot 13 showed homology with amphibian G1/S-specific cyclin E2, and spot 20 showed homology with the hypothetical protein DKFZp566A1524 of Brachidanio rerio. The present work shows that the use of the cryopreservation procedure causes the degradation of sperm proteins and among these, two could be at least partially responsible for the observed decrease in sperm motility duration and the lower hatching rate of eggs fertilized with cryopreserved sperm.

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The most common parameters used to evaluate sperm quality are motility rate and duration and fertilization ability. In this study, chemical and biochemical parameters of sea bass (Dicentrarchus labrax) sperm were investigated to find an alternative method for evaluating sperm fertilization ability before and after cryopreservation. The biochemical and chemical analyses were performed with fresh and frozen-thawed sperm and seminal plasma. To cryopreserve sperm, 250-microl straws were used. Fertilization ability was evaluated by inseminating eggs (obtained from hormonally stimulated females) with fresh and cryopreserved sperm. The results revealed a linear relationship (P < 0.05) between semen fertilization capacity and some seminal plasma (beta-D-glucuronidase activity, potassium concentration) and sperm (ATP concentration, aspartate aminotransferase activity) parameters. Variations in semen fertilization rate could be best described by two multiple regression models: one including the sperm parameters and another including the seminal plasma parameters. For practical application, the use of simple regression models is of value. Fertilization rate in both fresh and cryopreserved sperm was reliably predicted by determining the ATP concentration or the beta-D-glucuronidase activity or both.

In both herbivorous tilapia (Oreochromis mossambicus) and carnivorous rockfish (Sebastes caurinus) intestinal and pyloric cecal brush-border membrane vesicles (BBMV), [14C]glycylsarcosine ([14C]Gly-Sar) uptake was stimulated by a transmembrane proton gradient. A transmembrane K(+)-diffusion potential (inside negative) stimulated [14C]Gly-Sar uptake above that observed with short-circuited vesicles, whereas an inwardly directed Na+ gradient in both fishes had no effect on peptide uptake. In tilapia, [14C]Gly-Sar influx occurred by the combination of 1) a high-affinity, saturable, proton gradient-dependent carrier system [Kt [concentration that equals one-half of maximum influx (Jmax)] = 0.56 +/- 0.08 mM; Jmax = 1,945.0 +/- 174.6 pmol.mg protein-1.10 s-1]; 2) a low-affinity, nonsaturable (within 1-10 mM), proton gradient-dependent carrier system (nonsaturable carrier-mediated transport component = 4,514.0 +/- 28.1 pmol.mg protein-1.10 s-1.mM-1); and 3) a diffusional component accounting for < 10% of total influx within the concentration range tested. Influx (10 s) of 1-10 mM [14C]Gly-Sar in tilapia intestine was significantly (P < 0.01) inhibited by 10 mM diethylpyrocarbonate, a specific inhibitor of proton-coupled peptide transport systems. [14C]Gly-Sar influx into tilapia BBMV showed cis-inhibition and trans-stimulation by Gly-Pro, suggesting that [14C]Gly-Sar and Gly-Pro shared the same mucosal peptide transporter in fish. These observations strongly suggest that intestinal transport of peptides in herbivorous and carnivorous fishes is proton gradient dependent, electrogenic, sodium independent, and qualitatively resembles the peptide transport paradigm proposed for mammals.

D-[3H]glucose transport properties of brush-border membrane vesicles (BBMV) of upper intestine and pyloric ceca of the Pacific copper rockfish (Sebastes caurinus) were characterized and compared. Vesicles from both organs exhibited Na-dependent, phloridzin-sensitive, carrier-mediated transport systems. Kinetic constants for D-[3H]glucose influx across vesicle membranes were as follows: upper intestine, apparent affinity of glucose (Kt) = 0.14 +/- 0.02 mM, maximal glucose influx (JM) = 1,649 +/- 57 pmol.mg protein-1.10 s-1; pyloric ceca, Kt = 0.58 +/- 0.12 mM, JM = 2,439 +/- 178 pmol.mg protein-1.10 s-1. A hyperbolic relationship, following Michaelis-Menten kinetics, occurred between D-glucose influx and external Na concentration for pyloric ceca, while a sigmoidal function, following Hill cooperativity kinetics (n = 1.71 +/- 0.31), was disclosed between the variables for the intestine. External phloridzin, D-glucose, methyl alpha-D-glucopyranoside, and D-galactose were the most potent inhibitors of D-[3H]glucose influx in each organ. Other compounds were generally more inhibitory in vesicles from the pyloric cecum than those of the intestine except for D-mannose which was considerably more potent in the intestine. Results suggest that there may be proximal-to-distal hexose- and Na-binding gradients in the teleost gut for optimizing sugar absorption during passage of food through the gastrointestinal tract.