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Experiments were carried out to develop an optimal cryopreservation protocol for tench sperm by testing the fertilizing capacity and motility parameters including progressive motility, curvilinear velocity (VCL) and linearity (LIN) of cryopreserved sperm. Three experiments were designed to this aim: first experiment where we tested the effects of two extenders (sugar-based Grayling and ion-based Kurokura 180) and two cryoprotectants (DMSO and methanol) on fertilization and hatching success; second where we tested the effect of cryoprotectant type (methanol or DMSO) in different concentrations (5%, 10% and 15%) on fertilization and hatching success; and third where we tested the effect of two cryoprotectants (methanol and DMSO) on sperm motility parameters (progressive motility, VCL and LIN) after 4 h post-thaw storage (4°C). Sperm prepared with the sugar-based Grayling extender displayed better fertilization and hatching rates independently of the applied cryoprotectant most likely due to glucose present which acted as an external cryoprotectant. Concerning cryoprotectant concentrations, the use of 10% methanol yielded the highest fertilization (85 ± 15%) and hatching (80 ± 13%) rates, which were significantly higher than in all other groups. During the post-thaw storage time, 5% methanol, 10% methanol and 5% DMSO groups had significantly higher motility parameters than other groups and we observed no significant decline in any of the parameters during the storage time. Overall, we found that a sugar-based extender in combination with methanol as cryoprotectant is suitable for the cryopreservation of tench sperm and allows storage of cryopreserved sperm for up to 4 h post thaw.

Jundia (Rhamdia quelen) is an important species for aquaculture in temperate and subtropical climates. In the present study, the results of different forms of GnRH analogue treatments on ovulation in jundia have been compared with those from treatment with carp pituitary. Seven out of eight females ovulated in groups treated with carp pituitary (4.0 mg kg−1 BW) and Ovaprim (0.5 mL kg−1 BW). Injection with sGnRHa (10 μg kg−1 BW) combined with metoclopramide (20 mg kg−1 BW) caused a significantly lower rate of ovulation with three out of eight females responding. None of the fish injected with sGnRHa (10 μg kg−1 BW) alone ovulated. Mean PGSI [(weight of stripped egg mass/BW of the female before stripping) × 100] and mean fertilization rate values were high and similar between treatment groups. The obtained results indicate that in jundia, there is a strong dopamine inhibitory tone on gonadotropin secretion. The gonadotropin releasing activity of sGnRHa can be potentiated by dopamine receptor antagonists and the combined treatment is effective in inducing ovulation. In this regard, domperidone proved to be more potent than metoclopramide.

In our study, we used a full factorial analysis of variance design to examine the effects of diluent [Mounib's sucrose-based diluent+hen's egg yolk (EY) and Hanks' balanced salt solution (HBSS)+EY], freezing rate (−2.5, −5.0 and −7.5 °C min−1) and thawing rate (2.5, 5.0 and 7.5 °C min−1) on motility and velocity of Atlantic cod sperm cryopreserved in 2.5 mL cryogenic straws. We found that post-thaw sperm performance was strongly influenced by the presence of higher-order interactions of the factors we tested. For all models broken down by diluent, the 2.5 °C min−1 thawing rate had the lowest sperm motility recovery index. Mounib's sucrose-based diluent+EY had the highest motility recovery index at all thawing rates. Mean per cent motility for fresh sperm (87.7±2.9%) was not significantly different than of sperm cryopreserved using Mounib's sucrose-based diluent+EY, frozen at −2.5 °C min−1 and thawed at 5.0 °C min−1 (77.1±2.9%). For Mounib's sucrose-based diluent+EY, velocity was significantly higher with sperm thawed at 7.5 °C min−1, than sperm thawed at 2.5 °C min−1, while thawing rate had no effect for HBSS+EY. Our findings have implications for cod mariculture and aiding in conservation efforts for a dominant marine fish species.

The present study investigated motility, acrosome reaction, fertility and cryobiological parameters of the semen of the sterlet, Acipenser ruthenu sL . Sperm motility persisted for about 4 min in water, and the main swimming type was the linear motion. Motility was prolonged at osmolalities of 12.5 mosmol kg � 1 and in the presence of magnesium ions, while calcium had no eiect. Also a pH in the range of 7.0^9.0 had no eiect on ‘ motility. At osmolalities of 25^ 50 mosmol kg � 1 the sperm motility was partly inhibited, at osmolalities of 100 mosmol kg � 1 , completely and irreversibly. In 50 mosmol kg � 1 solutions with 2.5^5 mM L � 1 KCl the motility inhibition was total, but reversible. The acrosome reaction was not induced by one of the described solutions, but the percentage of spermatozoa with reacted acrosomes was low (o20%) and highly variable in all experiments. The optimal extender base for cryopreservation was a solution consisting of 50 mM L � 1 NaCl, 5m M L � 1 KCl, 10 mM L � 1 Tris (pH 8.5). From the tested cryoprotectants only dimethylsulphoxide (DMSO) and methanol provided su⁄cient cryoprotection. After freezing and thawing, the motility rates and swimming velocities were higher with DMSO than with methanol. However, the fertility was very signi¢cantly reduced with DMSO (10.3 � 0.5%) while with methanol fertilization rates in a similar range (32.7 � 4.4%) as with fresh semen (33.90 � 0.8%) could be obtained. Optimal freezing conditions for sterlet semen were in the vapour of liquid nitrogen 3^5 cm ( � 95 1 Ct o� 85 1C) above its surface, the optimal thawing conditions at 25 1 Cf or 30 s. The acrosome reaction was not induced by these cryopreservation protocols.

The effect of extenders was studied on the cryopreservation of sperm from African catfish, Clarias gariepinus (Burchell). The following six basic extenders were tested: fructose, glucose, sucrose, NaCl, KCl solutions and the artificial seminal plasma of the African catfish. Each of these extenders was tested both with and without buffer systems (i.e. NaHCO3-CO2 and Tris-HCl) by using 10% dimethyl-sulphoxide (DMSO) as a cryoprotectant. The two-step freezing was carried out in a programmable freezer by using the following freezing rates: (1) 4 °C min–1 between 3 and –4 °C; (2) and 11 °C min–1 between –4 and –80 °C. The best post-thaw motility (25%) was achieved with 333 mmol L–1 fructose solution and NaHCO3 buffer. The fertilization experiments were carried out with unbuffered fructose and glucose extenders using various amounts of sperm and two fertilization methods: (1) dry and (2) wet. The best fertilization rates were achieved with 75 μL of sperm and wet fertilization with glucose extender, or 100 μL of sperm and dry fertilization in case of fructose – both methods fertilized 96% of all eggs.