Giovanni Sansone

Effects of extender composition, cooling rate, and freezing on the motility of sea bass (Dicentrarchus labrax, L.) spermatozoa after thawing ☆

A successful cryopreservation procedure for sperm must guarantee recovery of the morphological and functional characteristics of the cells following thawing so that preserved semen can to be used comparably with non-preserved semen. The aim of this work was to identify a species-specific freezing protocol for sea bass (Dicentrarchus labrax) spermatozoa by optimising all the stages in the cryopreservation procedure. In the first stage of the experiments, the cryoprotectants and the relative concentrations that had the least toxic effect on motility at room temperature were selected. The capacity of the selected cryoprotectant substances was then assessed in freezing tests as follows: dimethyl sulfoxide (Me(2)SO) 5% and 7%, ethylene glycol (EG) 7% and 10%, propylene glycol (PG) 7% and 10%. The cryoprotectant that gave the best results in this second stage of the experiments was EG 10%, and this was then used for the optimisation of the different stages in the freezing procedure: two different times of adaptation to the cryoprotectant were tested (15min and 6h), as well as the effects of adding an energy substrate (1.25mM sodium pyruvate) to assess its possible use as an energy source. Lastly, using the extender (diluent+Na-pyruvate+EG10%) and the adaptation procedure (6h at 0-2 degrees C) that had given the best results in the preceding stages of the experiments, four cooling rates were tested: 10, 12, 15, 24 degrees C/min. It was shown that the semen that was diluted immediately after collection in extender that contained the cryoprotectant (EG 10%), was equilibrated for 6h at 0-2 degrees C and then cooled at a rate of 15 degrees C/min, showed motility on thawing comparable to that of fresh semen (P=0.045).

Cryopreservation of the Mediterranean mussel (Mytilus galloprovincialis) spermatozoa.

The effects of cryoprotectants, cooling rate and freezing on the mussel Mytilus galloprovincialis sperm were evaluated. At the end of each step of the experimental protocol, motility and fertilization ability of sperm were analyzed, compared to fresh semen. Five cryoprotectants were tested in their toxicity level: dimethylsulfoxide, ethylene glycol, 1-2 propylene glycol at 5%, 7%, 10%, 15% and 20% concentration; glycerol and methanol at concentration of 5%, 7% and 10%. The incubation times were 10, 20 and 30 min at 20+/-1 degrees C. Only dimethylsulfoxide, ethylene glycol and 1-2 propylene glycol at 5%, 7% and 10% were chosen for the following pre-freezing step. Five adaptation/chilling rates were analyzed: 10 min at 20+/-1, -2, -1, -0.5 and -0.25 degrees C/min and the last one was used for testing the best freezing procedure among seven gradients. Particularly, two rapid rates, three slow rates and two double step rates were conducted. Thawing results showed that M. galloprovincialis sperm are very sensitive to rapid pre-freezing and freezing protocols and only a slow procedure assured good motility and fertilization percentages.

Effects of cooling and freezing on the motility of Ostrea edulis (L., 1758) spermatozoa after thawing

The aim of this work was to evaluate the effects of temperature, cryoprotectant agents and freezing curves on sperm motility of Ostrea edulis. All phases of cryopreservation were studied (evaluation of semen motility pattern, choice of cryoprotectants and freezing rates) to restore after thawing the motility characteristics distinctive of fresh semen. To assess the temperature effects on sperm motility, semen was activated using four different temperatures (25, 18, 10 and 3°C). Sperm aliquots were maintained inactive at these temperatures for 1 and 3h, then activated with FSW at same temperature of conservation. Sperm was activated and incubated to 3°C with dimethylsulfoxide (Me(2)SO), ethylene glycol (EG), 1-2 propylene glycol (PG) (5%, 7%, 10% and 15% final concentrations), glycerol (GlOH; 5%, 10% and 15% final concentrations) and methanol (MetOH; 4% and 10% final concentrations) for 10, 20 and 30min. A first evaluation of freezing rates was made by testing four freezing curves: -1, -3, -6 and -10°C/min. Then, an optimization was made by testing four freezing curves: -2.5, -3.0, -3.5 and -4°C/min. The selected temperature for short term conservation has been 3°C, because only this temperature has allowed good sperm motility conservation after 3h of dry-storage; this is a time sufficient to conduct cryopreservation procedures. The sperm showed a particular sensitivity to GlOH and PG to all tested concentrations and to 15% Me(2)SO. EG and MetOH to all concentrations and Me(2)SO to concentrations lower than 15% have not shown significant toxic effects. The freezing rate -3°C/min using 15% EG has shown an highest percentage of RVF (rapid, vigorous and forward) spermatozoa (class 3, about 75% of fresh semen) and an highest sperm motility duration.

Cryopreserved semen in ecotoxicological bioassays: sensitivity and reliability of cryopreserved Sparus aurata spermatozoa.

The aim of this study was to evaluate the feasibility of using cryopreserved S. aurata semen in spermiotoxicity tests. Cryopreservation is a biotechnology that can provide viable gametes and embryos on demand, rather than only in the spawning season, thus overcoming a limitation that has hindered the use of some species in ecotoxicological bioassays. Firstly, the sperm motility pattern of cryopreserved semen was evaluated after thawing by means of both visual and computer-assisted analyses. Motility parameters in the cryopreserved semen did not change significantly in the first hour after thawing, meaning that they were maintained for long enough to enable their use in spermiotoxicity tests. In the second phase of the research, bioassays were performed, using cadmium as the reference toxicant, in order to evaluate the sensitivity of cryopreserved S. aurata semen to ecotoxicological contamination. The sensitivity of the sperm motility parameters used as endpoints (motility percentages and velocities) proved to be comparable to what has been recorded for the fresh semen of other aquatic species (LOECs from 0.02 to 0.03 mg L(-1)). The test showed good reliability and was found to be rapid and easy to perform, requiring only a small volume of the sample. Moreover, cryopreserved semen is easy to store and transfer and makes it possible to perform bioassays in different sites or at different times with the same batch of semen. The proposed bioassay is therefore a promising starting point for the development of toxicity tests that are increasingly tailored to the needs of ecotoxicology and environmental quality evaluation strategies.

Inactivator media of sea bass (Dicentrarchus labrax L.) spermatozoa motility

The spermatozoa of oviparous fish, such as sea bass, are immotile in the presence of semen plasma or solutions that are isotonic to it, and to obtain good motility, they must be diluted in a suitable medium like sea water. The object of this study was to identify the best protocol for the inactivation and activation of sea bass (Dicentrarchus labrax L.) spermatozoa motility in order to obtain an artificial inactivation of motility, which can be restored when necessary, so that semen can be used not only immediately after collection. Sea bass semen was obtained by abdominal stripping from 50 males in breeding during the reproductive season. The semen, kept at a temperature of 0–2 °C, was diluted with the experimental inactivator solutions DI1, DI2 and DI3. The solutions were tested at three ratios of dilution: 1:3, 1:6 and 1:10. The inactivated semen was activated by dilution at a ratio of 1:10 with sea water at pH 7.5 and 8.2. Subsequently, three different inactivated semen/sea water ratios of dilution were tested (1:3, 1:6, 1:10), using sea water at pH 8.2, which proved to have a better activating capacity. In addition, the motility resistance of inactivated, cooled (0–2 °C) semen was tested by activation every 12 h to evaluate its duration in time compared to that of undiluted semen. Motility was classified according to the percentage of rapid, vigorous and forward-moving motile spermatozoa (RVF). All tested solutions were good inactivators, as motility was always 0 after dilution. On activation, the best results were obtained with DI2 (dilution ratio 1:6), which restored motility in the highest percentage of spermatozoa; sea water pH 8.2 gave better results than sea water pH 7.5. Although no significant differences were found, a semen/inactivator medium of 1:6 dilution and an inactivated semen/activating solution of 1:10 seem to give the best results. Moreover, chilled (0–2 °C) semen diluted in DI2 medium preserved its motility longer than undiluted semen stored under the same conditions.

Biochemical characterization of the native α-carbonic anhydrase purified from the mantle of the Mediterranean mussel, Mytilus galloprovincialis

Abstract A α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified and characterized biochemically from the mollusk Mytilus galloprovincialis. As in most mollusks, this α-CA is involved in the biomineralization processes leading to the precipitation of calcium carbonate in the mussel shell. The new enzyme had a molecular weight of 50 kDa, which is roughly two times higher than that of a monomeric α-class enzyme. Thus, Mytilus galloprovincialis α-CA is either a dimer, or similar to the Tridacna gigas CA described earlier, may have two different CA domains in its polypeptide chain. The Mytilus galloprovincialis α-CA sequence contained the three His residues acting as zinc ligands and the gate-keeper residues present in all α-CAs (Glu106-Thr199), but had a Lys in position 64 and not a His as proton shuttling residue, being thus similar to the human isoform hCA III. This probably explains the relatively low catalytic activity of Mytilus galloprovincialis α-CA, with the following kinetic parameters for the CO2 hydration reaction: kcat = 4.1 × 105 s−1 and kcat/Km of 3.6 × 107 M−1 × s−1. The enzyme activity was poorly inhibited by the sulfonamide acetazolamide, with a KI of 380 nM. This study is one of the few describing in detail the biochemical characterization of a molluskan CA and may be useful for understanding in detail the phylogeny of these enzymes, their role in biocalcification processes and their potential use in the biomimetic capture of the CO2.

Binding of free and protein-associated zinc to rat spermatozoa

1. The zinc content of rat spermatozoa increases, upon ejaculation, from 0.035 to 1.055 micrograms/10(6) cells. 2. The rat seminal plasma holds zinc both as free ion and as protein-bound forms. 3. Zinc-free ions bind in vitro to rat epididymal spermatozoa. 4. Zinc-protein complexes can be isolated, by a chromatographic procedure, from the dorsolateral lobe of rat prostate. 5. The isolated zinc-protein complexes bind in vitro to rat epididymal spermatozoa.

Investigating sperm cryopreservation in a model tunicate, Ciona intestinalis sp. A.

In cryopreservation procedures, the capacity to protect the cells from freezing and thawing processes is sensitive to the choice of the cryoprotective agent (CPA) and to its optimal concentration. The advancement of research on Tunicate model species has raised interest in liquid nitrogen cryopreservation for the storage and distribution of genetic resources. Ciona intestinalis (Linnè, 1767) consists of a complex of cryptic taxa that are central to several areas of investigation, from comparative genomics to invasive biology. Here we investigated how five CPAs, three chilling rates and two freezing rates influence semen cryopreservation in C. intestinalis sp. A. By using larval morphology and motility as endpoints, we estimated that long term semen storage requires 10% dimethyl sulfoxide as a protective agent, -1°C/min chilling rate (18°C to 5°C) and -13°C/min freezing rate (5°C to -80°C), followed by immersion in liquid nitrogen.

Gamete cryobanks for laboratory research: Developing a rapid and easy-to-perform protocol for the cryopreservation of the sea urchin Paracentrotus lividus (Lmk, 1816) spermatozoa ☆

Gamete cryopreservation is a biotechnology that can guarantee a continuous supply of gametes, regardless of the seasonal reproductive cycle. In this study we developed a protocol for the cryopreservation of the sea urchin Paracentrotuslividus spermatozoa, with a view to the creation of cryobanks of semen to be used as a model system in laboratory research and ecotoxicological tests. All the key phases of the procedure were separately considered and the effect on sperm motility was evaluated by means of computer assisted analysis. The best results were obtained using 7% dimethylsulfoxide in 1% NaCl plus 0.04 M trehalose as the extender, at a freezing rate of -20 °C/min. On thawing, in semen samples cryopreserved in accordance with this protocol the velocity parameters of the sub-population of rapid sperm (best performing spermatozoa) did not significantly differ from semen on collection; in addition also the fertilization ability was restored, and about 50% of normal developed plutei larvae were obtained by thawed semen. The developed protocol is rapid and easy-to-perform; moreover, the use of gametes from reared urchins makes it unnecessary to continuously collect specimens from natural populations, making this procedure a promising starting point for the creation of alternative and more sustainable methodologies in laboratory research on sea urchin gametes and embryos.

Cloning, expression and purification of the α-carbonic anhydrase from the mantle of the Mediterranean mussel, Mytilus galloprovincialis

Abstract We cloned, expressed, purified, and determined the kinetic constants of the recombinant α-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the α-CA family is largely represented and plays a pivotal role in the deposition of calcium carbonate biominerals. Our results demonstrated that rec-MgaCA was a monomer with an apparent molecular weight of about 32 kDa. Moreover, the determined kinetic parameters for the CO2 hydration reaction were kcat =  4.2 × 105 s−1 and kcat/Km of 3.5 × 107 M−1 ×s−1. Curiously, the rec-MgaCA showed a very similar kinetic and acetazolamide inhibition features when compared to those of the native enzyme (MgaCA), which has a molecular weight of 50 kDa. Analysing the SDS-PAGE, the protonography, and the kinetic analysis performed on the native and recombinant enzyme, we hypothesised that probably the native MgaCA is a multidomain protein with a single CA domain at the N-terminus of the protein. This hypothesis is corroborated by the existence in mollusks of multidomain proteins with a hydratase activity. Among these proteins, nacrein is an example of α-CA multidomain proteins characterised by a single CA domain at the N-terminus part of the entire protein.