Spermatozoon ultrastructure and sperm cryopreservation of the Brazilian dry season spawner fish pirapitinga,Brycon nattereri

The aims of this study were to describe the fresh spermatozoon ultrastructure using scanning and transmission electron microscopy and to improve the sperm cryopreservation methodology for the freshwater fish pirapitinga Brycon nattereri. Extenders (BTS^? and NaCl), straw volumes (0.5 and 4.0 mL), thawing temperatures (30 and 60 °C) and activating agents (0.29% NaCl and 1% NaHCO₃) were tested. Methylglycol was used as a cryoprotectant agent and sperm was frozen in nitrogen vapour (dry‐shipper). Post‐thawed sperm motility rate, motility quality (score 0=no movement; 5=rapidly swimming spermatozoa), duration of motility and spermatozoon morphology were evaluated. Fresh spermatozoon was 35.06 μm long, the head was ovoid (2.00 × 1.22 μm) with no acrosome, the midpiece was 2.15 μm long and the flagellum was 30.90 μm long with the typical 9+2 axoneme arrangement. Post‐thawed sperm motility rate (70–79% motile sperm), motility quality (score 3.1–3.7) and morphology (9.3–11.6% abnormal spermatozoa) were not affected by any of the parameters tested. The duration of sperm motility was longer when triggered in 1% NaHCO₃ (392–1031 s) compared with 0.29% NaCl (144–338 s). Brycon nattereri sperm cryopreserved under the conditions described above yields over 70% motility and should last long enough to fertilize oocytes, even after 2 years of freezing.     

Sperm motility in the steelhead Oncorhynchus mykiss (Walbaum): influence of the composition of the incubation and activation media

This study examined the pH sensitivity of steelhead, Oncorhynchus mykiss (Walbaum), sperm motility relative to the composition of incubation and activation media. The percentage of sperm that initiated motility following incubation in a sperm immobilizing solution (SI) titrated to different pH values and subsequent activation by dilution in buffered swimming medium (SM) at pH 8.5 or 50% ovarian fluid (OF) showed little or no pH sensitivity; sperm diluted in de‐ionized water (DI) showed no motility after incubation at any pH. In contrast, motility of sperm diluted in tap water (TAP) was highly sensitive to the pH of the incubation medium. Sperm incubated with buffered seminal plasma at high, but not low pH demonstrated high percent motility when diluted with DI. Sperm incubated in low‐pH SI demonstrated high motility only when diluted into high‐pH SM. The effects of the composition of incubation and activation media on sperm motility were generally reflected in comparable effects on fertility. Therefore, these data indicate that the pH sensitivity of sperm motility and fertility depends on the composition of commonly used incubation as well as activation media.     

Early ontogeny of yellowtail tetra fish Astyanax lacustris (Characiformes: Characidae)

The ontogeny of the tetra fish Astyanax lacustris, from embryogenesis through the larval and juvenile periods, is presented. The eggs and larvae were obtained through induced reproduction. The larvae were collected at hatching and daily, until all stages of development were obtained and, later, once a week, until the juvenile period. The eggs are small, slightly adhesive and demersal, and have a transparent chorion, yellowish calf and meroblastic cleavage. The larvae hatch 19 hr after fertilization (26ºC). The standard length during initial development ranges from 2.17 to 18.10 mm. The larvae have a subterminal mouth, simple nostrils, a pair of adhesive organs on the back of the head (newborn larvae), spherical eyes and medium‐length intestines. The initial pigmentation is scarce, concentrated in the anteroposterior ends of the yolk sac, but intensifies from the flexion stage, with chromatophores in the mouth and in the cephalic region; the humeral region and the caudal peduncle have a macula at the end of the larval development. The total number of myomeres: 32–38, 15–20 preanal and 14–20 postanal. Juveniles showed similar morphology and pigmentation to adults. The number of fin rays: pectoral 11–12; pelvic 6–7; dorsal 10–11; and anal 27–29.     

Sperm fertility of the subtropical freshwater streaked prochilod Prochilodus lineatus (Characiformes) improved after dilution and cold storage

The aims of this study were to evaluate the efficiency of simple and complex extenders in prolonging the cold storage of sperm (Experiments 1 and 2) and to test the diluted‐cooled sperm in the best extender with regard to sperm quality parameters (Experiment 3) in the streaked prochilod, Prochilodus lineatus. In all the experiments, aliquots of 0.3 mL of sperm were diluted 1:10 in extenders and stored at 4–6 °C. Sperm diluted in simple extenders (NaCl and glucose solutions) yielded 0–26% sperm motility, whereas sperm diluted in complex extenders (BTS^?, M III^? and Androstar^?) yielded 62–81% sperm motility on day 4 after cold storage. When Androstar^? was further investigated, the following was observed on day 4: 53% motility with 94 s of duration; 47% live spermatozoa; 26–61% fertility rate; and 22–60% hatching rate. The use of Androstar^? improves the sperm fertility of the streaked prochilod during a 4‐day storage period and can therefore be used to facilitate artificial reproduction.     

抗冻剂对日本鳗鲡精子活力及运动时间的影响

利用精子稀释液(0.5%氯化钠+0.25%氯化钾+0.25%葡萄糖)分别配制4%、8%、12%、16%、20%、24%的二甲亚砜(DMSO)、甘油(Glycerol)、1,2-丙二醇(1,2-Propanediol)、乙二醇(Ethyleneglycol)四种抗冻剂,对日本鳗鲡精子进行激活处理,观察记录精子在不同浓度抗冻剂中的活力和5种运动(激烈运动、快速运动、慢速运动、摆动、死亡)时间。结果表明:4%~24%的四种抗冻剂都可以激活日本鳗鲡精子,其中二甲亚砜浓度为12%时,精子活力最高,平均可达到78.3%;甘油浓度为8%时,精子活力最高,平均可达到72.8%;12%的1,2-丙二醇和乙二醇激活精子后,精子活力都可以达到最高,分别是47.6%和57.8%。日本鳗鲡精子在8%的二甲亚砜、8%的甘油、8%的1,2-丙二醇和8%的乙二醇中,激烈运动时间均为最长,分别为11.8 s、11.1 s、6.0 s和2.9 s;用8%的甘油、16%的二甲亚砜、16%的1,2-丙二醇和12%的乙二醇激活日本鳗鲡精子后,精子存活时间均最长,分别达到154.6 s、45 s、20 s和34 s。与其它几种抗冻剂相比,8%的甘油能显著延长日本鳗鲡精子的寿命。四种抗冻剂对日本鳗鲡精子都有一定的毒性作用,处理后精子的活力和寿命均降低。     

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.     

Sperm cryopreservation of silver barb (Barbodes gonionotus): cryoprotectants,cooling rate and storage time on sperm quality

Effectiveness and efficiency of frozen sperm on fertilization and hatching success of eggs from silver barb was examined in relation to cryoprotectants, freezing rate and storage period. Sperm was diluted in calcium‐free Hank's balanced salt solution, equilibrated with dimethylsulphoxide (DMSO), propylene glycol, sucrose or methanol at 5%, 10%, 15% or 20% final concentrations, and frozen in 250‐μL straws using a one‐step freezing procedure (1, 5 and 8°C min^?1 from 25 to ?40°C). Highest post‐thaw sperm motility was found from a treatment using 10% DMSO and 5°C min^?1 (82.2 ± 2.1%), similar to that of 10% DMSO and 8°C min^?1 (87.8 ± 3.2%). Post‐thaw motility of sperm frozen at 5 or 8°C min^?1 was significantly higher than 1°C min^?1. Relative sperm motility declined significantly after 10 months of cryostorage while viability did not change during a 12‐month cryostorage. Average fertilization rates of sperm after 1 and 4 months of storage were 64.5 ± 4.6% and 61.3 ± 3.4%, respectively, similar to those of fresh sperm (69.6–72.3%). Hatching rates of cryopreserved sperm (45.4–51.2%) were similar to those of fresh sperm (51.8–57.8%). This study developed suitable methods for cryopreservation of silver barb sperm that can be used to facilitate hatchery operation.     

Development of cryopreservation methods for silver barb (Barbodes gonionotus,Bleeker) spermatozoa by manipulation of cooling rates in dry shipper

The aim of this study was to develop a simple cryopreservation protocol for silver barb, Barbodes gonionotus, semen using a dry shipper. Freezing rates within the upper and lower chambers of dry shipper were recorded for 14 days post liquid nitrogen loading (dpl). To regulate freezing rates, straws (250 and 500 µl) wrapped with various insulators (polystyrene foam box, oxygen tube, silicone tube and electric wire) were frozen within the upper chamber. Straws containing semen diluted with Calcium‐free Hank's Balanced Salt Solution (Ca‐F HBSS) and 10% dimethyl sulphoxide were cryopreserved with or without insulators. Appropriate protocols were selected based on sperm quality during a 45‐day cryostorage. The upper chamber had potential as a freezing chamber within 9 dpl due to no significant (p > 0.05) change in freezing rates. High percentages of sperm motility and viability (p < 0.05) were observed when 250 µl straws with silicone tube (T4) frozen for 5 min, non‐insulated 500 µl straws (T9) and 500 µl straws with polystyrene foam box (T12) frozen for 1–5 min, having freezing rates of 43.1 ± 1.3, 71.3 ± 1.4 and 14.7 ± 0.4°C/min respectively. Dry shipper can be used as a freezing tool to cryopreserve silver barb semen.     

Effect of extender composition and freezing rate on post-thaw motility and fertility of Arctic char, Salvelinus alpinus (L.), spermatozoa

The effects of extender composition and freezing rate on motility and fertility of frozen‐thawed Arctic char, Salvelinus alpinus, spermatozoa were investigated. Three freezing rates, two semen diluents and three cryoprotectants were tested. Semen frozen in 0.3 mol L^?1 glucose diluent with 10% methanol as a cryoprotectant or in a diluent described by Lahnsteiner with 10%N,N‐dimethylacetamide (DMA) resulted in the highest sperm motility. Fertility was the highest for semen frozen in a glucose–methanol extender but was not significantly different than that for semen frozen in Lahnsteiner's diluent with 10% DMA. Dimethyl sulphoxide (DMSO) at 10% was a relatively ineffective cryoprotectant with either semen diluent. Semen frozen at 6 cm above the surface of liquid nitrogen resulted in a higher post‐thaw sperm motility and fertility than semen frozen at 5 cm. The addition of 7% fresh egg yolk to glucose diluent containing methanol or DMSO did not improve the fertility of frozen‐thawed spermatozoa. However, the addition of 7% fresh egg yolk to glucose–DMA extender significantly improved the fertilization percentages of frozen‐thawed spermatozoa. In conclusion, dilution of semen 1:3 in 0.3 mol L^?1 glucose with 10% methanol and freezing 6 cm above the surface of liquid nitrogen (freezing rate of 40±8°C min^?1, mean±SD from ?5 to ?55°C) is a promising protocol for cryopreservation of Arctic char semen.     

Sperm motility and fertilization performance of Nodipecten nodosus (L., 1758) exposed at two different cryoprotectants

Cryopreservation is a valuable tool for aquaculture as it provides continuous seed production, regardless of the spawning season of the brood stock. The selection of a suitable cryoprotectant with low toxicity and high water solubility is important to avoid membrane injuries and intracellular ice crystallization. This study has been aimed at the assessment of the toxic effects of two usually applied cryoprotectants, 1‐2 propylene glycol (PG) and methanol (MetOH), on spermatozoa of the of lion‐paw scallop Nodipecten nodosus, by evaluating the sperm motility and the development of D larvae after fertilization procedure. Sperm was exposed at room temperature (22°C) for 10, 20 and 30 min to different concentration ranges of two cryoprotectants. Regarding the sperm motility, PG5%, PG7%, MetOH4% and MetOH6% did not show differences compared to control (semen incubated in seawater) (P < 0.05). The development of D larvae was not affected by the exposition to PG5%, MetOH 4% and MetOH 6%. These results indicate the potential use of both cryoprotectants for cryopreservation procedures.     

Toxic effects of mercury chloride on silver catfish (Rhamdia quelen) spermatozoa

Heavy metals are highly toxic elements that are present in the environment, especially in water. Mercury chloride (HgCl₂) stands out among these compounds because of its strong ability to induce damage to any tissue with which it comes into contact. The gametes of spawning aquatic animals, such as fish, are susceptible to such damage. Thus, our objective was to evaluate the toxic potential of HgCl₂ in the capacitation and activation of Rhamdia quelen sperm. Semen was collected from seven males and activated in 58 mM sodium chloride (NaCl) containing 0 (control), 4^?10, 7^?10, 7^?9, and 7^?8 M HgCl₂. The evaluated variables included motility, vigor, motility time, morphology, membrane integrity, membrane fluidity, mitochondrial functionality, production of reactive oxygen species (ROS), and DNA fragmentation. All evaluated HgCl₂ concentrations increased primary pathologies and reduced motility, vigor and motility time. Damage to membrane integrity and fluidity began occurring at a concentration of 7^?10 M HgCl₂. These results indicate that HgCl₂ has a toxic effect on different sites of fish spermatozoa and that sperm motility decreases after exposure to HgCl₂, impairing sperm capacitation and activation.     

Effect of Dimethyl sulfoxide (DMSO) and egg yolk on sperm motility,fertility and hatching rates of depik Rasbora tawarensis (Pisces: Cyprinidae) eggs after short‐term cryopreservation

Cryoprotectant is the crucial factor in the cryopreservation process. In general, there are two types of cryoprotectant, permeating and non‐permeating cryoprotectants. Dimethyl sulfoxide (DMSO) and egg yolk are common permeating and non‐permeating cryoprotectants respectively. Hence, the objective of the present study was to determine the best proportion of DMSO and egg yolk for the cryopreservation of Rasbora tawarensis sperm. A completely randomized experimental design was used in this study which involves two types of cryoprotectant and their combination at different concentrations, namely 5% DMSO, 5% egg yolk, 5% DMSO + 5% egg yolk and 2.5% DMSO + 2.5% egg yolk. Every treatment was conducted in three replicates. Combination of 5% DMSO + 5% egg yolk gave the best results cryoprotectant treatment had significant effects on sperm motility, fertilization and hatching rate of the R. tawarensis eggs (p < .05). It is concluded that the best proportion of cryoprotectants for sperm cryopreservation in this species is 5% DMSO + 5% egg yolk.     

环境因子对胭脂鱼精子活力影响的研究

通过观察精子的快速运动时间和寿命,研究了6种环境因子对胭脂鱼(Myxocyprinus asiaticus Bleeker)精子活力的影响。结果表明:胭脂鱼精子对酸碱的耐受能力较强,在pH值为4~10时,精子均可以快速运动,且在pH值为8时精子的活力最强。不同浓度的NaCl、KCl、葡萄糖、CaCl2、MaCl2溶液对胭脂鱼精子运动的诱导效果不同。5种溶液最适于胭脂鱼精子运动的浓度分别为:NaCl 102 mmol/L;KCl 75 mmol/L;葡萄糖100 mmol/L;CaCl275.6 mmol/L;MgCl275.6 mmol/L。且在5种溶液低浓度时精子活力较强,超过一定浓度精子的运动开始被抑制。     

Comparative gonad histology and semen quality of normal (XY) and neo‐males (XX) of Atlantic salmon (Salmo salar)

With an overarching objective of improving the hatchery production of Atlantic salmon (Salmo salar) all‐female progeny, this study comparatively evaluated the reproductive parameters between normal (genotype XY) and neo‐males (genotype XX). Four normal (XY) and seven neo‐ (XX) males, from the same brood stock, distinguished by their ability to or lack of expressing semen, respectively, were comparatively evaluated. The left testicular lobe was used for histomorphometric analyses, while the right for semen collection and sperm quality analyses. Histomorphometric observations revealed that neo‐male testes are irregularly shaped, and have poorly formed seminiferous ducts, higher proportions of interstitial tissue and lower gonadosomatic index (p < .05). In addition, hypertrophied and cyst forming Sertoli cells were found in these individuals which collectively appear to form a physical barrier, precluding the semen collection by standard stripping techniques and reducing sperm quality. Particularly, semen motility (80.69 ± 2.4% and 57.2 ± 36.5% for XY and XX respectively) and duration of motility (99.31 ± 28.03 s and 66.84 ± 23.83 s for XY and XX respectively) of neo‐males were most compromised (p < .05). Interestingly, the TUNEL assay indicated no signs of apoptotic tissue suggesting that the histological differences may relate to delayed physiological/sexual maturity of neo‐males.     

不同盐度激活液、K+浓度及保存时间对日本鳗鲡精子活力的影响

在6种不同盐度(34、32、30、28、26和22)激活液、3种不同K+浓度(25 mmol/L、30 mmol/L和35 mmol/L)稀释液和不同保存时间(0 h、24 h、48 h、72 h和96 h)条件下,对日本鳗鲡(Anguilla japonica)精子的活力进行观察和测定.结果表明,激活液盐度为30时精...     

日本黄姑鱼精子生理特性及超低温冷冻保存研究

对日本黄姑鱼精子部分生理特性及其超低温冷冻保存技术进行了研究。结果表明,日本黄姑鱼精液pH值为7.0~7.5,精子密度为(11.23±2.78)×109/m l,精子寿命(229.33±17.16)s。在盐度为30~35,pH为7.5~8.5时,精子的活力最高,分别达到(88.33±2.89)%和(88±4.33)%。精子在室温(25℃)条件下,可存活24 h;在低温(4℃)条件下可以存活36 h。以D液作为稀释液,20%的乙二醇作为抗冻剂,利用快速降温法对精子进行超低温冷冻保存,38℃水浴快速解冻,解冻后的精子获得最高的活力为(47±5.78)%。     

超低温冷冻对俄罗斯鲟精子顶体酶活性及DNA损伤的影响

选取6 ind健康的雄性俄罗斯鲟(Acipenser gueldenstaedti),经人工催产后获得成熟的精子,研究超低温冷冻(-196℃)对俄罗斯鲟精子顶体酶活性及DNA损伤影响。结果显示:俄罗斯鲟鲜精中顶体酶的平均活性为(36.18±2.54)μIU·10-6,经过超低温冷冻后,精子顶体酶活性显著降低,添加抗冻保护液精子中顶体酶活性降至(21.55±0.79)μIU·10-6,未添加抗冻保护液精子中顶体酶活性降至(9.58±1.08)μIU·10-6,且三者间有显著性差异(P0.05)。单细胞凝胶电泳结果表明,俄罗斯鲟鲜精彗星率为(37.33±7.77)%,添加抗冻剂后冻精的彗星率为(63.67±5.13)%,未添加抗冻剂直接冷冻彗星率高达(86.00±3.61)%,三者间有显著差异(P0.05)。用CASP分析软件分析测量彗星拖尾长度(L tail)、彗星尾部DNA的相对含量(Tail DNA)、尾动量(TM)、Olive尾动量(OTM)等各项表征DNA损伤的指标,发现冻精组的各项指标均显著高于鲜精组,未添加抗冻剂直接冷冻组又高于添加抗冻剂组,3组间有显著性差异(P0.05)。本研究结果表明:超低温冷冻能导致精子顶体酶活性下降和DNA损伤,抗冻剂对精子具有保护作用。     

Effect of cryoprotectants, equilibration periods and freezing rates on cryopreservation of spermatozoa of mahseer, Tor khudree (Sykes) and T. putitora (Hamilton)

A study was conducted to standardize a protocol for cryopreservation of spermatozoa of the endangered mahseer, Tor khudree (Sykes) and T. putitora (Hamilton). The suitability of the cryoprotectants, dimethyl sulphoxide (DMSO) and glycerol, and the combination of the two were tested. Four equilibration periods and four freezing rates were also tested for their standardization. A combination of 9% DMSO and 11% glycerol gave significantly higher mean percentage of hatching in both T. khudree (45.59±1.86%) (control 71.08±0.59%) and T. putitora (45.00±1.25%) (control 73.48±1.19%) among the eight different treatments. Among the four different equilibration periods tested, the equilibration period of 30 min^?1 yielded the highest mean hatching percentage in T. khudree (39.46±1.94%) (control 71.70±0.61%) and T. putitora (38.28±1.06%) (control 73.11±0.82%). Freezing straws at a height of 8 cm above LN₂ surface for 10 min^?1 gave higher hatching percentages for both T. khudree (41.75±1.72%) (control 73.99±1.17%) and T. putitora (41.34±2.04%) (control 72.48±1.51%). The study reports the superior performance of the combination of DMSO and glycerol for the first time.     

Use of MS‐222 (tricaine methanesulfonate) and propofol (2,6‐diisopropylphenol) as anaesthetics for the tetra Astyanax altiparanae (Teleostei,Characidae)

The aim of this study was to evaluate the anaesthetic effect of MS‐222 and propofol and determine their optimal concentrations for safe handling of the tetra Astyanax altiparanae in the laboratory. The fish were separated by length into three classes: I (1.5–5.0 cm), II (5.1–8.0 cm) and III (greater than 8.1 cm). Pilot tests were performed to evaluate the appropriate anaesthetic concentrations for inducing the five possible anaesthetic stages: I – sedation; II – light anaesthesia; III – deep anaesthesia; IV – surgical anaesthesia; and V – spinal collapse. After defining the maximum and minimum concentrations required to induce stage IV anaesthesia, the animals were exposed to five intermediate concentrations (n = 10 fish/concentration) of each anaesthetic for 15 min. The animals were then transferred to clean water to evaluate the time required for recovery. In addition, blood glucose levels were measured for class II and class III fish subjected to the previously defined ideal concentrations for each of the tested anaesthetics (n = 10 fish/treatment). Both evaluated substances are suitable to anaesthetize A. altiparanae. The optimal MS‐222 concentration was 90 mg L^?1, and this result was similar for all three size classes. The optimal propofol concentrations for inducing surgical anaesthesia in the size classes I, II and III were 0.22, 0.23 and 0.27 respectively.