Motility of sea urchin Paracentrotus lividus spermatozoa in the post‐activation phase

The characterization of sperm motility patterns, particularly post‐activation changes, is the first step in setting up species‐specific protocols involving gamete management and embryo production, for both aquaculture and laboratory research purposes. This study is aimed at the characterization of the sperm motility pattern of the purple sea urchin Paracentrotus lividus. Semen samples were individually diluted in artificial sea water for sperm motility activation. They were then incubated at 18°C for up to 24 hr. Motility was evaluated on dilution, and 1 hr, 3 hr and 24 hr after activation, by computerized analyser. The semen fertilization capacity was also evaluated. Under our experimental conditions (dilution 1:1,000 in artificial sea water plus 0.05% BSA, 18°C, in the dark), P. lividus semen remained viable for up to 24 hr, as the total motile sperm and the fertilization percentages did not change significantly during the incubation time. In contrast, the mean curvilinear velocity and the subpopulation of rapid sperm (those having a curvilinear velocity > 100 µm/s) slightly but significantly decreased after 3 hr, thereafter remaining unchanged for up to 24 hr after activation. In conclusion, our results show that diluted P. lividus semen can be used for a longer period than that of most fish species, with no need for motility inhibition procedures, supporting its wider use in laboratory research. In addition, the development of artificial fertilization protocols for aquaculture production is simplified by long‐lasting sperm motility.     

Cryopreservation of Persian sturgeon (Acipenser persicus) sperm: effects of cryoprotectants,antioxidant, membrane stabilizer,equilibration time and dilution ratio on sperm motility and fertility

Three experiments were performed to develop protocols for cryopreservation of Persian sturgeon Acipenser persicus, sperm. In the first experiment, sperm from six males was individually split in three subsamples and cryopreserved using Modified Tsvetkova's extender (mT) supplemented with dimethyl sulfoxide (DMSO), methanol (MeOH), glycerol (Gly) and ethylene glycol (EG) at concentration of 5%, 10%, 15% and 20%. In the second set of experiments, the effects of six equilibration times (0, 5, 10, 20, 40 and 60 min) and dilution ratios (volume sperm: volume extender 1:0.5, 1:1, 1:2, 1:3, 1:5 and 1:10) and the additive advantage of bovine serum albumin (BSA; 0, 2.5, 5 and 10 mg mL^?1) and ascorbic acid (0, 2.5, 5 and 10 U mL^?1), on the post‐thaw survival of sperm (triplicate set of six fish) were evaluated. Then, sperm was diluted in 1:1 mT extender with 10 mg mL^?1 BSA with selected cryoprotectants (15% MeOH and 10% DMSO) for 5 min. After a month of storage in liquid nitrogen, post‐thawed sperm motility; fertilization and hatching rate and viability of derived larvae were measured (Exp.3). Evaluation of cryoprotectants efficiency showed that MeOH 15% and DMSO 10% were suitable for cryopreservation of Persian sturgeon sperm. Gly and EG resulted in very low post‐thaw motility rates even at lowest concentration. No significant difference was observed among the four different equilibration times (0, 5, 10, 20 min) (P > 0.05) although higher equilibration times than 20 min resulted low post‐thaw motility (P < 0.05). The motility of frozen–thawed sperm did not significantly change when dilution ratio was increased from 1:0.5 to 1:3 (P > 0.05). However, higher dilution ratios (1:5 and 1:10) reduced the percentage of motile sperm. Supplementation of the cryoprotectant solution with 10 mg mL^?1 BSA significantly improved post‐thaw motility (P < 0.05), but ascorbic acid did not improve post‐thaw motility (P > 0.05). The results of experiment 3 showed that the highest fertilization (30.2 ± 5.75) and hatching rates (28.2 ± 5.25) were observed when samples were frozen with 15% MeOH (P > 0.05). Our study indicates that the use of mT extender consisting of 10 mg mL^?1 BSA in 15% MeOH diluted with sperm at 1:1 ratio for 5 min can be recommended cryopreservation method for Persian sturgeon sperm.     

Relationships between body size and secondary sexual characters,and sperm characters in male Dolly Varden char (Salvelinus malma)

In Salmonidae, subordinate males are exposed to higher risks of sperm competition than dominant males and thus are expected to improve the sperm characteristics (sperm concentrations, sperm velocity and sperm longevity). In this study, we investigated the relationships between body size and secondary sexual characters (breeding colour, hump height and snout length), and sperm characteristics of one‐year‐old (newly matured) Dolly Varden char. Small males displayed higher sperm concentrations than large males. Moreover, males with dull breeding colours, but not with lesser snout length and hump height, displayed an increased sperm velocity compared to males with bright colours, suggesting a trade‐off between sperm quantity and the investment in breeding colour. In addition, sperm longevity decreased as sperm swimming velocity increased. These findings indicate that small males with dull breeding colours improve the quantity and quality of their sperm to a great extent to enhance their chances of reproductive success.     

诱导栉孔扇贝雌核发育时精子入卵的扫描电镜观察

取栉孔扇贝 (Chlamysfarreri)精子在 80 0 μW/(cm2 ·s)的紫外线下辐射 ,然后与正常卵子受精 ,每隔 0 .5min取“受精卵”固定。在扫描电镜下连续观察精子的入卵过程 ,然后与正常精子的入卵过程进行比较。结果表明 ,紫外辐射后的精子可以划分为 3种类型 :I类精子 ,基本无明显变化 ,可以激发卵黄膜举起形成受精膜 ,同时诱发卵子发生皮层反应 ,在受精锥的作用下正常入卵 ,但入卵的时间较正常精子晚 ;II类精子 ,顶体膜破裂 ,顶体丝伸出 ;III类精子 ,顶体丝伸出 ,鞭毛脱落。由于II、III类精子在紫外辐射条件下诱发了顶体反应 ,融解卵膜的酶提前释放 ,因而不能入卵。精子鞭毛的脱落 ,使精子丧失了运动能力 ,是导致卵表面附着的精子数量较少的原因之一。无论是正常精子还是辐射后的精子 ,入卵后在卵表面都留下一个类似受精孔的通道。实验组中未发现有多精入卵现象     

Fertility and motility of sperm from Atlantic halibut (Hippoglossus hippoglossus) in relation to dose and timing of gonadotrophin-releasing hormone agonist implant

In broodstocks of Atlantic halibut, Hippoglossus hippoglossus, male and female gamete production often becomes unsynchronised towards the end of the spawning season—milt becomes very viscous and difficult to express while the females are still producing batches of good quality eggs. Gonadotrophin-releasing hormone agonist (GnRHa) has been shown to stimulate spermiation in a number of fish species. Therefore, we conducted two experiments where male halibut were implanted intramuscularly with pellets containing GnRHa. The effect of the pellets was tested at three periods: before, at the height of and at the end of spermiation. In the middle period, GnRHa was tested at two doses (5 and 25 μg/kg bodyweight). Measurements were made of milt hydration, sperm motility and fertilisation rate. Implanted males began spermiation at least 4 weeks before control males. Both doses of GnRHa increased the fluidity of the milt. This effect lasted for at least 20 days in the low dose group and for 40 days in the high dose group. When applied at the end of the season, GnRHa reversed the normal trend for the milt to become more viscous. GnRHa treatments did not affect fertilisation rates obtained with the sperm. However, towards the end of the spawning season, sperm motility was enhanced in males treated with the high dose of GnRHa (25 μg/kg) compared to controls. As described previously, plasma concentrations of the gonadal steroids, 5β-pregnane-3β,17,20β-triol 20-sulphate and 17,20-dihydroxy-4-pregnen-3-one, were significantly enhanced by GnRHa treatment. Concentrations of testosterone on the other hand decreased when spermiating males were treated with GnRHa. Our data suggest that 17,20β-dihydroxy-4-pregnen-3-one or its metabolites are involved in milt hydration, possibly through affecting ion transport.     

Physio-Chemical Characteristics of Seminal Plasma and Development of Media and Methods for the Cryopreservation of European eel Sperm

The high sperm density, together with the short spermatozoa swimming time, makes European eel sperm manipulation and assessment for quality difficult. Two diluting media (K15 and K30) previously designed for Japanese eel sperm were tested. After 24 h, European eel sperm showed significant reduction in the percentage of motile spermatozoa after activation and different motility parameters (VAP, angular velocity; VCL, curvilinear velocity; VSL, straight line velocity; BCF, beating cross frequency), concluding that these media are not suitable to preserve the sperm of this species. After a hormonal treatment to induce spermiation, sperm volume, density and motility were recorded at weekly samplings. The variation of the osmolality (325–330 mOsm kg⁻¹), pH (8.4–8.6) and the ionic composition (concentration of Na⁺, K⁺, Mg²⁺ and Ca²⁺) of the seminal plasma were registered. Physio-chemical results were related with sperm quality throughout the treatment, to determine which must be the suitable characteristics of one extender for the sperm of this species, and to find the best conditions to obtain suitable cryopreservation media for European eel sperm. K⁺ concentration increased, while Ca²⁺ and Mg²⁺ concentrations showed a progressive reduction in correlation with the sperm quality improvement. Na⁺ showed a decreasing, but not significant tendency. P1 and P2 freezing media were designed considering the physio-chemical parameters as well as the ionic composition shown by the best quality sperm samples, and then compared with the previously described solutions, TNK and K30. Sperm quality was determined, checking the percentage of motile spermatozoa and motility parameters using computer-assisted sperm analysis (CASA) software. Samples were frozen after dilution (1:5, 1:20, 1:100) in different freezing media supplemented with 10% dimethyl sulfoxide (DMSO). After thawing, samples frozen with low dilution ratio (1:5) in TNK and P1 media showed higher, although not significant, spermatozoa survival (35.5 ± 14.5 and 36.6 ± 6.7%). The addition of l-α-phosphatidylcholine to the media seems to have a positive effect, as reported in the Japanese eel.     

The Ionic and Osmotic Factors Controlling Motility of Fish Spermatozoa

This review presents actual knowledge about energetic, ionic, osmotic and gaseous control of fish sperm motility and its duration. Right after they are activated, fish spermatozoa of most species swim for a short period of time, in the range of one to several minutes. What determines the activation process? Is it due to the new ionic, gaseous and/or osmotic environment? Why is the duration of motility so short? Is it resulting from a fast exhaustion of energy stores (ATP, ADP, AMP, PCr) combined with the above-mentioned ionic/osmotic stress leading to morphological alterations? The motility criteria (flagellar beat frequency, head displacement velocity, flagellar waves morphology, etc.) used to characterize fish sperm movement and sperm flagella will be described. Most parameters change very rapidly during the brief motility period of fish sperm. Then will be considered the main environmental factors, ionic and/or osmotic signals, responsible of the activation of fish sperm motility. Then the metabolic compounds involved in cell energetics will be considered as their concentrations also rapidly change during the motility phase. An additional feature will then be discussed concerning the mechanisms by which fish sperm cell can be revived for a second motility round at the end of the first motility period. A model is proposed to explain how external osmolarity can control internal ionic composition, the latter being the key factor controlling flagellar activity.     

Role of sodium bicarbonate on the initiation of sperm motility in the Japanese eel

ABSTRACT:   In order to find out the role of sodium bicarbonate (NaHCO₃) on the initiation of sperm motility in the Japanese eel Anguilla japonica , interactions were investigated between NaHCO₃ and various reagents (K⁺ channel blocker 4-aminopyridine [4-AP], ammonium chloride [NH₄Cl], sodium acetate and calcium chloride [CaCl₂]) that could regulate internal factors (intracellular K⁺, intracellular pH [[pH]_i] and intracellular Ca²⁺) in sperm motility. Contradictory effects of NaHCO₃ were observed (i.e. an inhibitory effect when 4-AP was absent and a promoting effect when 4-AP was present). Sodium bicarbonate inhibited the initiation of sperm motility in the Japanese eel. However, NaHCO₃ restored the motility of immotile sperm that 4-AP inhibited. The inhibitory effect of NaHCO₃ disappeared with the addition of NH₄Cl, which raised [pH]_i, but the promoting effect was not affected by [pH]_i. Although NaHCO₃ recovered motility in the presence of 4-AP, this recovery was also observed with the addition of CaCl₂ instead of NaHCO₃. In the initiation of sperm motility in the Japanese eel, two roles for NaHCO₃ are suggested: an inhibitory role relating to the regulation of [pH]_i and a promoting role relating to the uptake of another initiation factor, which could be Ca²⁺.